DNA Extraction PDF

DoD DNA Registry
Office of the Armed Forces Medical Examiner

Armed Forces Institute of Pathology
Washington, DC 20306-6000
ARMED FORCES DNA IDENTIFICATION LABORATORY
DNA EXTRACTION MANUAL
Version 1.0
Adopted By: ______________________________________ Date: _______________
Reviewed By: ______________________________________ Date: _______________
Page 1 of 70
TABLE OF CONTENTS
CHELEX® EXTRACTIONS...................................................................................................................................... 3
I. PRINCIPLE .................................................................................................................................................... 3
II. BIOLOGICAL SPECIMENS ......................................................................................................................... 3
III GENERAL REAGENTS, SUPPLIES AND EQUIPMENT .......................................................................... 3
IV. QUALITY ASSURANCE.............................................................................................................................. 4
V. SAFETY ......................................................................................................................................................... 5
VI. PROCEDURES & WORKSHEETS – CHELEX EXTRACTIONS................................................................. 6
A. Chelex Extraction of DNA from Whole Blood and Bloodstains – Procedure ............................................ 7
B. Chelex® Extraction of DNA from Tissue and Fresh Bone - Procedure...................................................... 9
C. Chelex Extraction of DNA from Swabs – Procedure ............................................................................... 11
D. Chelex Extraction of DNA from Post-Coital Specimens - Procedure...................................................... 14
E. Chelex Extraction of DNA from Tissue Embedded in Paraffin - Procedure............................................ 17
VII. REFERENCES ........................................................................................................................................ 20
ORGANIC EXTRACTIONS ................................................................................................................................... 21
I. PRINCIPLE .................................................................................................................................................. 21
II. BIOLOGICAL SPECIMENS ....................................................................................................................... 21
III. GENERAL REAGENTS, SUPPLIES AND EQUIPMENT ........................................................................ 21
IV. QUALITY ASSURANCE............................................................................................................................ 22
V. SAFETY ....................................................................................................................................................... 23
VI. PROCEDURES & WORKSHEETS – ORGANIC EXTRACTIONS ............................................................ 25
A. Organic Extraction of DNA from Whole Blood – Procedure .................................................................. 26
B. Organic Extraction of DNA from Bloodstains – Procedure .................................................................... 28
C. Organic Extraction of DNA from Tissue – Procedure ............................................................................. 32
D. Organic Extraction of DNA from Fresh Bone – Procedure..................................................................... 35
E. Organic Extraction of DNA from Swabs – Procedure ............................................................................. 38
F. Organic Extraction of DNA from Hair Roots – Procedure...................................................................... 41
G. Organic Extraction of DNA from Hair Shafts – Procedure..................................................................... 45
H. Organic Extraction of DNA From Dried Skeletal Remains – Procedure ................................................ 52
I. Organic Extraction of DNA from Teeth – Procedure .............................................................................. 56
J. Organic Extraction of DNA from Human Nail Material – Procedure..................................................... 61
K. Organic Extraction of DNA from Urine – Procedure.............................................................................. 65
VII. REFERENCES ........................................................................................................................................ 68
VIII. ADMINISTRATIVE REVIEW ............................................................................................................... 70
Page 2 of 70
CHELEX® EXTRACTIONS
I. PRINCIPLE
Chelex® 100 is a chelating resin composed of styrene divinylbenzene copolymers containing

paired iminodiacetate ions, which act as chelating groups. The presence of Chelex during
boiling prevents the degradation of DNA by chelating metal ions that may otherwise catalyze the
breakdown of DNA subjected to high temperatures. The basic procedure consists of a wash to
remove some of the contaminants such as heme and proteins, followed by boiling the sample in a
5% Chelex solution.
NOTE: When pipetting Chelex solutions, the resin beads must be distributed evenly in
solution. This can be done by gentle mixing with a stir bar in a beaker. Use a pipet tip
with a large bore (200 -1000 µl).
NOTE: DNA extracted with Chelex is single-stranded and is unsuitable for quantitation
methods that use intercalating agents such as ethidium bromide.
II. BIOLOGICAL SPECIMENS
The first part of this manual contains standard operating procedures and corresponding
worksheets for the extraction of DNA, using Chelex 100, from the following biological
specimens:
Whole Blood and Bloodstains

Tissue and Fresh Bone
Swabs (e.g. oral)
Post-Coital Specimens
Tissue Embedded in Paraffin
Cytological Slides
III GENERAL REAGENTS, SUPPLIES AND EQUIPMENT
Chelex 100® (Bio-Rad) in sterile, deionized water. Make fresh daily.

Beaker
Bleach, 10% commercial (7mM sodium hypochlorite solution)
Boiling Water Bath
Ethanol
Forceps
Freezer, -20oC
Gloves
Heat Block
Hole Puncher (1/8 inch)
Incubator (56oC and 37°)
Kim-wipes (no-lint type recommended for certain steps)
Laboratory coat
Magnetic Stirring Plate
Microcentrifuge (e.g., Eppendorf)
Pipettors (e.g., P-10, P-100, P-1000, PipetAid)
Page 3 of 70
Racks, tube
Refrigerator, 4oC
Safety glasses
Scalpels
Scissors
Stir bar
Tips, aerosol-resistant (e.g., for P-10, P-100, P-1000 pipettors)
Tubes, (1.7 ml)
Ultraviolet Crosslinker
Vortex
Waste containers (general, biohazard, SharpsTM)
Water, sterile, distilled
Weigh Boats
*Special supplies, reagents and equipment for procedures will be listed within the
procedure itself.
IV. QUALITY ASSURANCE
1. Extraction of specimens is to be performed in the following designated laboratories:
whole blood specimens and bloodstains Lab 3

specimens suspected of containing high levels of DNA Lab 4
2. Ensure that all reagents, instruments, and equipment satisfy the minimum standards for
quality control, where appropriate.
3. Chelex should be made fresh daily. For a 5% solution, add 0.5 g Chelex to 10 ml of
dH2O.
4. Any laboratory workspace and all pipettors and racks to be used in this procedure must
be cleaned with 10% commercial bleach and thoroughly dried before beginning. When
using a laminar flow hood, expose to ultraviolet light for a minimum of 10 minutes after
cleaning.
5. Before use, laboratory instruments must be cleaned with 10% commercial bleach and
dried thoroughly (i.e. pipettes). In addition, they should be cleaned in the same manner
following the processing of each individual specimen.
6. Microcentrifuge tubes (closed) should be irradiated in an ultraviolet crosslinker with 2

J/cm2. Follow "Irradiation of Reagents and Supplies in the Ultraviolet Crosslinker" SOP
for appropriate method of irradiation.
7. No reagents used for the extraction of DNA from specimens are allowed in a post-
amplification room.
8. Any supplies or equipment taken from a post-amplification room to a pre-amplification

room must be sterilized with 10% commercial bleach before removal from the post-
amplification room and again in the pre-amplification room before use.
Page 4 of 70
9. Only one evidence specimen should be open at any one time. For CILHI cases, only
evidence specimens from one case should be extracted together. For OAFME cases and
databasing, more than one case can be extracted at a time.
10. A minimum of one reagent blank should be carried throughout the extraction procedure
and assayed in parallel with the evidence samples. If available, a substrate control should
be carried throughout the extraction and assayed in parallel with the evidence. Order of
processing for each procedure is as follows: substrate control(s), specimen(s), reagent
blank(s).
11. Change pipet tips between each transfer or addition of sample or reagent, unless
otherwise noted.
12. No aliquot of any reagent may be returned to the original stock container.
13. Use aerosol-resistant pipet tips for all liquid transfers.
V. SAFETY
1. All appropriate MSDS sheets must be read prior to performing this procedure.
2. Treat all biological specimens as potentially infectious. Gloves, safety glasses, and a
laboratory coat must be worn at all times.
3. Follow the "Exposure Control Plan for Occupational Exposure to Bloodborne Pathogens"
(DoD Forensic Advisory Committee).
4. Avoid direct exposure to ultraviolet light when using the germicidal lamp in the
biological hood or the transilluminator.
5. The hot plate can become very hot. Be careful not to touch the heating surfaces while in
operation.
6. Use proper protective equipment to prevent burns when handling boiling water or hot
solutions.
7. Distinguish all waste as general, biohazard, organic, chemical or SharpsTM and discard
appropriately.
Page 5 of 70
VI. PROCEDURES & WORKSHEETS – CHELEX EXTRACTIONS
Page 6 of 70
A. Chelex Extraction of DNA from Whole Blood and Bloodstains – Procedure
1. Label 1.7 ml microcentrifuge tubes with a minimum of AFDIL case number and
specimen number.
2. Pipette 1 ml of sterile, deionized water into the 1.7 ml microcentrifuge tubes. A reagent
blank must be initiated at this point.
3. Add 3 - 5 µl of whole blood or a piece of bloodstained material (approximately a 1/8"

diameter punch or a 3 mm square) to the tube.
4. Vortex at high speed and incubate at room temperature for a minimum of 15 minutes.
Note: Vortex tubes several times during incubation to dislodge heme.
5. Spin in microcentrifuge for 3 minutes at 10,000 - 15,000 rpm.
6. Discard all but 20 - 30 µl of the supernatant. Leave substrate and pelleted material in the
tube.
7. Resuspend the pellet in 5% Chelex to a final volume of 200 µl.
8. Incubate at 56o C for at least 30 minutes.
9. Vortex at high speed for 10 seconds.
10. Incubate in a boiling water bath for 8 minutes
12. Spin in microcentrifuge for 3 minutes at approximately 10,000 - 15,000 rpm.
13. The sample is now ready for quantitation (optional) and amplification.
14. Place at 2-8˚C on the Chelex beads for short-term storage. To use after short-term
storage on the beads, repeat steps 10 (optional), 11 and 12. For long-term storage,
transfer the supernatant from the Chelex beads to a sterile, labeled tube and freeze.
You will need the following worksheet for this procedure:
Chelex Extraction of DNA from Whole Blood and Bloodstains – DNA Form 4
Page 7 of 70
Chelex® Extraction of DNA from Whole Blood and Bloodstains
Followed Version _____ of the "AFDIL DNA Extraction Manual”
Case Number:_________________________Date:____________________Scientist:_________________________
Item Source Lot #
Sterile Water
Chelex®-100 AFDIL QC
Specimen Type Amount
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Reagent Blank #:
1. Add 1 ml sterile, distilled water to microcentrifuge tube.

2. Add specimen to tube (3-5 µl whole blood or 1/8" bloodstain punch).
3. Vortex at high speed and incubate at room temperature for a minimum of 15 minutes.
4. Spin at 10,000-15,000 rpm for 3 minutes in a microcentrifuge.
5. Discard all but 20-30
®
µl of the supernatant. Leave substrate in tube.
6. Add 5% Chelex -100 to a volume of 200 µl.
7. Incubate at 56oC for at least 30 minutes.
8. Vortex at high speed for approximately 10 seconds.
9. Incubate in a boiling water bath for 8 minutes.
10. Vortex at high speed for approximately 10 seconds.
11. Spin at 10,000-15,000 rpm for 3 minutes in a microcentrifuge.
Comments: ____________________________________________________________________________________
______________________________________________________________________________________
Location of DNA Extracts __________________________________________________________________________
DNA Form 4 Revised 022601
Page 8 of 70
B. Chelex® Extraction of DNA from Tissue and Fresh Bone - Procedure
1. Label 1.7 ml microcentrifuge tubes with a minimum of AFDIL case number and
specimen number.
2. Dissect a piece of tissue or bone approximately 1 - 2 mm square.
3. Add the tissue or fresh bone to 200 µl of 5% Chelex in a 1.7 ml microcentrifuge tube.
4. Incubate at 56o C for 30 minutes.
5. Vortex at high speed for 5 - 10 seconds.
7. Vortex at high speed for 5 - 10 seconds.
8. Spin in microcentrifuge for 3 minutes at 10,000 rpm.
10. Place at 2-8˚C on the Chelex beads for short-term storage. To use after short-term
storage on the beads, repeat steps 6 (optional), 7 and 8. For long-term storage, transfer
the supernatant from the Chelex beads to a sterile, labeled tube and freeze.
Chelex Extraction of DNA from Tissue and Fresh Bone – DNA Form 5
Page 9 of 70
Chelex® Extraction of DNA from Tissue and Fresh Skeletal Remains
Item Source Lot #
Sterile Water
Chelex®-100 AFDIL QC
Specimen Type Amount
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Reagent Blank #:
1. Add 200 µl of 5% Chelex®-100 to a 1.5 ml tube.

2. Add dissected piece of tissue or skeletal remains to tube (approximately 1-2 mm2 square).
3. Incubate at 56oC for 30 minutes.
4. Vortex at high speed for 5-10 seconds.
7. Microcentrifuge for 3 minutes at 10,000-15,000 RPM.
Comments: ____________________________________________________________________________________
_________________________________________________________________________________________________
Location of DNA Extracts __________________________________________________________________________
Page 10 of 70
C. Chelex Extraction of DNA from Swabs – Procedure
Special Supplies: Spin-EASE™ Tubes. If no Spin-EASE tubes are available, a 0.5 ml thin-wall
tube with a hole pierced into the bottom inside a 1.7 ml tube can be substituted (piggy-
back spin).
0.5 ml tube with hole = basket portion of Spin-EASE
1.7 ml tube = lower portion of Spin-EASE
Dried swabs and dried stains:
1. Dried swabs proceed to step 3.
2. Dried stains need to be collected on a sterile swab. Moisten a sterile swab with sterile water.
Swab the stained area and allow the swab to air dry in a laminar flow hood. Initiate a
substrate control.
Note: For histological slides place several drops (500ul -1000ul) of the appropriate
mounting media solvent (100% xylene dissolves Permount; toluene dissolves Cytoseal)
around the perimeter of the coverslip in order to remove the coverslip.
3. Using a fresh blade or clean scissors, cut half the swab off the applicator stick and place into
the lower portion of a Spin-EASE tube labeled (at a minimum) with AFDIL Case Number
and Specimen Number. (If it is suspected that an insufficient amount of material is present on
the swab, use the entire swab.) When using a cotton swab, the cotton should be teased with
forceps and/or a scalpel before transfer to tube.
4. Add 1.0 ml sterile water to the tube.
6. Incubate at room temperature for 30 minutes.
7. Centrifuge for 15 minutes at 12,000 - 15,000 rpm. Remove and discard top 0.5 ml of
supernatant.
8. Transfer the swab to the sterile, basket portion of a Spin-EASE extraction tube with forceps
or a clean pipet tip. Place the basket into the lower portion of the Spin-EASE tube. Spin for
3 minutes at 12,000 - 15,000 rpm. Proceed to step 15.
9. Label the Spin-EASE basket and place in a weigh boat in fume hood to allow the swab to air
dry overnight. When dried, repackage and store as evidence.
Swabs preserved in isopropanol:
10. Using a fresh blade or clean scissors, cut half the swab off the applicator stick and place into
a 15 ml conical tube.
11. Add 5 ml sterile water to the tube. A reagent blank must be initiated at this point.
Page 11 of 70
14. Centrifuge for 15 minutes at 2000 rpm. Remove and discard top 4.5 ml of supernatant.
15. Press excess liquid out of the swab and perform a piggy back spin. Perform piggy-back spin
as follows: Transfer the swab to the sterile, basket portion of a Spin-EASETM extraction tube
(or to a 0.5 ml thin-wall tube with a hole pierced into the bottom). Transfer the remaining
liquid and debris to the lower portion of the Spin-EASETM tube (or 1.7 ml). Place the basket
(or 0.5 ml tube) into the lower portion of the Spin-EASETM tube (or 1.7 ml). Spin for 3
minutes at 12,000 - 15,000 rpm.
16. Label the Spin-EASETM basket (or 0.5 ml tube) and place in a weigh boat in fume hood to
allow the swab to air dry overnight. When dried, repackage and store as evidence.
17. Remove and discard all but 50 µl of supernatant.
18. Add 5% Chelex to a final volume of 200 µl. Mix gently to resuspend the pellet.
NOTE: When pipetting Chelex solutions the resin beads must be

distributed evenly in solution. This can be done by gentle mixing with a
stir bar in a beaker. Use a pipet tip with a large bore (200 -1000 µl).
19. Incubate at 56˚ C for 15 - 30 minutes.
20. Vortex briefly.
22. Vortex briefly.
23. Spin in microcentrifuge for 3 minutes at 12,000 - 15,000 rpm.
25. Place at 2-8˚C on the Chelex beads for short-term storage. To use after short-term storage on
the beads, repeat steps 12 (optional), 13 and 14. For long-term storage, transfer the
supernatant from the Chelex beads to a sterile, labeled tube and freeze.

Chelex Extraction of DNA from Swabs – DNA Form 226
Page 12 of 70
Chelex® Extraction of DNA from Swabs
Followed Version____of the “AFDIL DNA Extraction Manual”
Case Number: __________________________ Date:_____________ Scientist:________________
Item Source Lot #

Sterile Water
Chelex 100 AFDIL QC
Specimen Amount Extracted Comments

Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Reagent Blank #
1. Cut the swab in half (or use whole swab if necessary) and place in the lower portion of a Spin-EASE tube.
2. Add 1 ml sterile water.
5. Centrifuge for 15 minutes at 12,000-15,000 rpm. Remove and discard top 0.5 ml of supernatant.
6. Transfer swab to the basket portion of Spin-EASE tube. Replace the lower portion of the Spin-EASE tube. Centrifuge for 15 minutes at
12,000-15,000 rpm.
7. Place the Spin-EASE tube aside and allow the swab to air dry overnight. When dried, repackage and store as evidence.
8. Remove and discard all but 50 µl of supernatant.
9. Add 5% Chelex solution to a final volume of 200 µl.
10. Incubate at 56°C for 15-30 minutes.
11. Vortex briefly. Incubate in a boiling water bath for 8 minutes.
12. Vortex briefly. Centrifuge for 3 minutes at 12,000-15,000 rpm.
Location of DNA Extracts____________________________________

Page 13 of 70
D. Chelex Extraction of DNA from Post-Coital Specimens - Procedure
Special Supplies: Proteinase K (20 mg/ml), 1 M Dithiothreitol (DTT), Extraction buffer (10mM
Tris, pH 8.0; 100 mM NaCl; 50 mM EDTA, pH 8.0; 0.5% SDS), and Spin-EASE™
Tubes. If no Spin-EASE tubes are available, a 0.5 ml thin-wall tube with a hole pierced
into the bottom inside a 1.7 ml tube can be substituted (piggy-back spin).
1. Label the lower portion of an appropriate number of Spin- EASE extraction tubes as well
as 1.7 ml microcentrifuge tubes.
2. Add 800 µl sterile, distilled water into each labeled Spin- EASE extraction tube.
3. Using clean scissors or a sterile scalpel blade, dissect the swab or fabric cutting on a clean
surface. Add the specimen (½ of the swab, or approximately 3 mm square of fabric
cutting) to the tube.
4. Vortex briefly and incubate at room temperature for 30 minutes.
5. Vortex for 10 seconds to agitate the cells off the substrate. Pulse spin.
6. Transfer the substrate to the basket of the Spin-Ease™ tube with either forceps or a pipette
tip. Place the basket containing the substrate into the lower portion of the Spin-Ease unit.
7. Spin the sample in a microcentrifuge for 1 minute at maximum speed (10,000-

15,000 rpm).
8. Remove the substrate to a clean, labeled tube. (Retain the swab or fabric for any future
analyses).
9. Without disturbing the pellet, remove and discard all but approximately 50 µl of the
supernatant. Resuspend the cellular debris pellet by stirring with a pipette tip.
NOTE: This pellet contains both epithelial and sperm cells.
10. Add 150 µl sterile distilled water and 2 µl of Proteinase K (20 mg/mL) to the resuspended
cellular debris pellet and mix gently.
11. Incubate at 37˚C for approximately 1 hour to lyse epithelial cells, but for no more than 2
hours to minimize lysis of sperm cells.
12. Spin in microcentrifuge for 5 minutes at 10,000- 15,000 rpm.
13. Without disturbing the sperm pellet, transfer 150 µl of the supernatant to a new 1.7 mL
microcentrifuge tube, labeled “E” (Epithelial, or female fraction). Add 50 µl of 20%
Page 14 of 70
Chelex solution (2 g Chelex in 10ml of distilled water). Store at room temperature until
Step 17.
14. Resuspend the pellet in 0.5 mL extraction buffer. Vortex briefly. Spin in a
microcentrifuge for 5 minutes at 10,000-15,000 rpm. Remove and discard all but 50 µl of
the supernatant. Repeat twice for a total of three washes.
15. Add 150 µl of 5% Chelex, 2 µl of Proteinase K (20 mg/mL) and 7 µl of 1 M dithiothreitol

(DTT) to the sperm pellet and mix gently. This is the male, or sperm fraction of the stain
and should be labeled with an “S”.
16. After the final buffer wash, add 1 ml of sterile distilled water to the pellet. Vortex briefly
and spin in a microcentrifuge for 5 minutes at 10,000 rpm. Remove and discard all but 50
µl of the supernatant.
17. Incubate at 37˚C for 30-60 minutes.
18. Vortex the epithelial (E) and sperm (S) cell samples at high speed for 5-10 seconds.
19. Spin in a microcentrifuge for 10-20 seconds at 10,000-15,000 rpm.
20. Incubate the samples in a boiling water bath for 8 minutes.
21. Vortex tubes for 5-10 seconds.
22. Spin in a microcentrifuge for 3 minutes at 10,000-15,000 rpm.
23. The samples are now ready for quantitation (optional) and amplification.
24. Place at 2-8˚C on the Chelex beads for short-term storage. To use after short-term storage
on the beads, repeat steps 20 (optional), 21 and 22. For long-term storage, transfer the
Chelex Extraction of DNA from Post-Coital Specimens – DNA Form 40
Page 15 of 70
Chelex® Extraction of DNA from Post-Coital Specimens
Case Number: __________________________ Date:_________________ _______Scientist:____________________________
Item Source Lot #

Sterile Water
Chelex 100 AFDIL QC
Specimen Amount Extracted Comments

Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Reagent Blank #
1. Add 800 µl sterile, distilled water into each labelled Spin- EASE™ extraction tube.
2. Using clean scissors or a sterile scalpel blade, dissect the swab or fabric cutting on a clean surface. Add the specimen to the
tube.
3. Vortex briefly and incubate at room temperature for 30 minutes.
4. Vortex for 10 seconds to agitate the cells off the substrate. Spin briefly.
5. Transfer the substrate to the basket of the Spin-EASE tube with either forceps or a pipette tip. Place the filter unit containing
the substrate into the appropriate lower portion.
6. Spin the sample in a microcentrifuge for 1 minute at maximum speed (10,000-15,000 rpm).
7. Remove the substrate to a clean, labelled tube. (Retain the swab or fabric for any future analyses). Remove and discard all but
approximately 50 µl of the supernatant. Resuspend the cellular debris pellet by stirring with a pipette tip.
8. Add 150 µl sterile distilled water and 2 µl of Proteinase K (20 mg/mL) to the resuspended cellular debris pellet and mix
gently. Incubate at 37°C for up to 1 hour but for no more than 2 hours.
9. Spin in microcentrifuge for 5 minutes at 10,000-15,000 rpm.
10. Transfer 150 µl of the supernatant to a new 1.7 mL microcentrifuge tube, labelled “E” (Epithelial, or female fraction). Add 50
µl of 20% Chelex solution. Store at room temperature until Step 13.
11. Wash the sperm pellet according to protocol.
12. Add 150 µl of 5% Chelex, 2 µl of Proteinase K (20 mg/mL) and 7 µl of 1 M dithiothreitol (DTT) to the sperm pellet and mix
gently. This is the male, or sperm fraction of the stain and should be labelled with an “S”.
13. Incubate at 37°C for 30-60 minutes.
14. Vortex the epithelial (E) and sperm (S) cell samples at high speed for 5-10 seconds.
15. Spin in a microcentrifuge for 10-20 seconds at 10,000-15,000 rpm.
16. Incubate the samples in a boiling water bath for 8 minutes.
17. Vortex tubes for 5-10 seconds.
18. Spin in a microcentrifuge for 3 minutes at 10,000-15,000 rpm.

Page 16 of 70
E. Chelex Extraction of DNA from Tissue Embedded in Paraffin - Procedure
Special Reagents, Supplies and Equipment: Absolute Ethanol, Xylene, Tissue Lysis Buffer,
Centricon 100™ concentrators, IEC MP4 Centrifuge, Microtome or scalpel, Hetovac.
Perform in a Laminar Flow Hood
1. If appropriate, cut away the outer layer of the paraffin block with a sterile blade or a
microtome.
2. Label tubes with a minimum of AFDIL Case Number and Specimen Number. Obtain two
thin slices of tissue specimen (approximately 10µm thick) using a sterile blade or a
microtome. Alternatively, a small section of tissue (approximately 2-4 mm thick) can be
taken from the block using a sterile blade. Place the tissue in an appropriately labeled
(AFDIL Case Number and Specimen number), sterile 1.7 ml microcentrifuge tube. Initiate
a substrate control at this time.
Perform in a Chemical Fume Hood
3. Add 1 ml xylene to each tube (including the reagent blank and the substrate control) to
remove the paraffin wax.
4. Incubate for 30 minutes at room temperature.
5. Spin for 2 - 5 minutes at 12,000 rpm.
6. Remove and discard the supernatant into the appropriate waste container.
7. Repeat steps 3 through 6 for a total of 2 xylene washes.
8. Add 1 ml absolute ethanol to each tube. Spin for 2 minutes at 12,000 rpm.
9. Remove and discard the ethanol into the appropriate waste container.
10. Repeat steps 8 and 9 for a total of 2 ethanol washes.
11. Allow tissue to vacuum dry in the Hetovac, approximately 10 minutes.
12. Prepare Tissue Lysis Buffer (fresh daily - see below). Add 200 µl of Tissue Lysis Buffer
to each tube.
Tissue Lysis Buffer:
1.89 ml TE Buffer (10mM, pH 7.5)
10 µl Tween 20 (0.5%)
100 µl Proteinase K (20mg/ml)
Total Volume 2.00 ml
Page 17 of 70
13. Incubate at 37o C overnight.
14. Spin for 5 minutes at 12,000 rpm.
15. Prepare the appropriate number of properly labeled Centricon-100 concentrators.
16. Remove supernatant and transfer to the labeled Centricon-100 concentrator. Discard any
remaining tissue sample in the appropriate biohazard container.
17. Spin 30-60 minutes, or longer if necessary (until liquid has passed through concentrator),
at 1000 x g using the IEC MP4 Centrifuge (2600 RPM). Discard filtrate.
18. Add 2 ml TE buffer to the sample reservoir. Spin 30-60 minutes, or longer if necessary, at
1000 x g using the IEC MP4 Centrifuge (2600 RPM). Discard filtrate. Repeat once.
19. Transfer the supernatant directly from the Centricon-100 to a properly labeled 1.7 ml
microcentrifuge tube.
20. Cut the pipet tip for a P100 pipettor in order to increase the size of the bore. Add 20 µl of
the 5% Chelex solution to the above tube.
21. Vortex briefly and incubate at 56˚ C for 30 minutes.
25. Spin in a microcentrifuge for 3 minutes at 12,000 rpm.
26. The samples are now ready for quantitation (optional) and amplification.
27. Place at 2-8˚C on the Chelex beads for short-term storage. To use after short-term storage
on the beads, repeat steps 23 (optional), 24 and 25. For long-term storage, transfer the
Chelex Extraction of DNA from Tissue Embedded in Paraffin – DNA Form 22
Page 18 of 70
Extraction of DNA from Tissue Embedded in Paraffin
Followed Version ______ of the "AFDIL DNA Extraction Manual”
Case Number: ________________________ Date: _______________ Scientist: ___________________
Extraction
Item Source Lot #
Xylene AFDIL QC
Ethanol AFDIL QC
TE Buffer AFDIL QC
Tween 20 AFDIL QC
Proteinase-K AFDIL QC
Centricon 100 Amicon
Chelex® AFDIL QC
Centricon Purification
# Specimen Approximate Approximate Comments
Volume Recovered (in µl) Final Volume (in µl)
Specimen #
Specimen #
Specimen #
Specimen #
Specimen #
Reagent Blank #
1. Place samples into the appropriately labeled 1.7 ml microcentrifuge tube.
2. Add 1 ml xylene.
3. Incubate 30 minutes at room temperature.
4. Spin for 2-5 minutes at 12,000 rpm.
5. Remove and discard the supernatant into the appropriate waste container .
6. Repeat steps 2-5 one time.
7. Add 1 ml absolute ethanol.
9. Remove and discard the supernatant into the appropriate waste container.
10. Repeat steps 7-9 one time.
11. Vacuum dry in the Hetovac, approximately 10 minutes.
12. Add 200 µl of Tissue Lysis Buffer.
13. Incubate overnight at 37°C.
15. Remove supernatant and transfer to the Centricon-100 concentrator.
16. Spin for 30 - 60 minutes at 1000 x g (2600 rpm) using the IEC.
17. Add 2 ml TE Buffer and spin for 30 - 60 minutes. Repeat once.
18. Remove supernatant and transfer to the appropriately labeled 1.7 ml microcentrifuge tube.
19. Add 20 µl of a 5% Chelex solution. Vortex briefly. Incubate at 56°C for 30 minutes
20. Vortex briefly. Incubate in a boiling water bath for 5 minutes.
21. Vortex briefly. Spin for 3 minutes at 12,000 rpm .
Location of DNA Extracts __________________________________________________
Page 19 of 70
VII. REFERENCES
Bio-Rad Laboratories Catalog (1996) Bio-Rad Laboratories, Hercules, CA, p. 85.
Bio-Rad Laboratories Chelex 100 and Chelex 20 chelating ion exchange resin instruction manual
(1996), pp. 1-24.
Comey, C. T., et.al. (1994) “DNA Extraction Strategies for Amplified Fragment Length
Polymorphism Analysis,” Journal of Forensic Sciences 39: 1254-1269.
Forsthoefel K.F., Papp A.C., Snyder P.J., Prior T.W., (1992) “Optimization of DNA Extraction
from Formalin-Fixed Tissue and its Clinical Application in Duchenne Muscular Dystrophy,”
A.J.C.P. 98(1): 98-104.
Gill P., Kimpton C., Sullivan K., (1992) “A rapid polymerase chain reaction method for
identifying fixed specimens”, Electrophoresis 13:173-175.
Heller M.J., Burgart L.J., TenEyck C.J., Anderson M.E., Greiner T.C., Robinson R.A., (1991)
“An Efficient Method for the Extraction of DNA from Formalin-Fixed, Paraffin-Embedded
Tissue by Sonication”, Biotechniques 11(3): 372-377.
Walsh, P. Sean, Metzger David A. and Higuchi, Russell, (1991) “Chelex® 100 as a Medium for
Simple Extraction of DNA for PCR-Based Typing from Forensic Material”, Biotechniques
10(4): 506-513.
Willard, J.M., Lee, D.A., Holland, M.M., “Recovery of DNA for PCR amplification from blood
and forensic samples using a chelating resin,” DNA Profiling Protocols in Methods in Molecular
Biology, 98(1): 9-18.
Page 20 of 70
ORGANIC EXTRACTIONS
I. PRINCIPLE
To provide instructions for isolating DNA from several sources using organic solvents. The
basic procedure consists of lysing nucleated cells with sodium dodecyl sulfate (SDS) coupled
with protein digestion with proteinase K. After digestion is complete, a series of
phenol/chloroform/isoamyl alcohol extractions is performed followed by a butanol rinse. The
DNA is purified and recovered by Centricon® 30 or 100 or by ethanol precipitation.
II. BIOLOGICAL SPECIMENS
The second part of this manual contains standard operating procedures and corresponding
worksheets for the extraction of DNA, using organic solvents, from the following biological
specimens:
Whole Blood
Bloodstains
Soft Tissue
Fresh Bone
Swabs
Hair Roots
Hair Shafts
Dried Skeletal Remains
Teeth
Human Nail Material
Stamps and Envelopes
Urine
III. GENERAL REAGENTS, SUPPLIES AND EQUIPMENT
Bleach, 10% commercial (7mM sodium hypochlorite solution)

Centricon-100® and/or Centricon-30® Concentrators
Centrifuge (MP4)
Ethanol, Absolute
Extraction buffer (10mM Tris, pH 8.0, 100 mM NaCl, 50 mM EDTA, pH 8.0, 0.5% SDS)
Forceps
Freezer, -20oC
Gloves {Viton™ or 6 mil (or greater) nitrile required for certain steps}
Incubator (37oC)
Incubator (56oC)
Kim-wipes (no-lint type recommended for certain steps)
Laboratory coat
Microcentrifuge (e.g., Eppendorf)
n-Butanol
Page 21 of 70
Nutator
Phenol/Chloroform/Isoamyl Alcohol (25:24:1)
Proteinase K (10mg/ml) or Proteinase K (20mg/ml)
Pipettors (e.g., P-10, P-100, P-1000, PipetAid)
Racks, tube
Refrigerator, 4oC
Safety glasses
Scissors
Sodium dodecyl sulfate (SDS)
Sodium Acetate
SSC (1X)
TE Buffer (10 mM Tris, 1mM EDTA, pH 7.5)
Tips, aerosol-resistant (e.g., for P-10, P-100, P-1000 pipettors)
Tubes, (1.7 ml, 15 ml)
Ultraviolet crosslinker
Vortex
Waste containers (general, biohazard, SharpsTM)
Water, sterile, distilled
Weigh boats
*Special supplies, reagents and equipment for certain procedures are listed at the
beginning of that procedure.
IV. QUALITY ASSURANCE
1. Extraction of specimens is to be performed in the following designated laboratories:
whole blood specimens and bloodstains Lab 3

specimens suspected of containing high levels of DNA Labs 4 and 5
specimens suspected of containing low levels of DNA Labs 9 and 10
Note: Specimens that are dry (i.e., no fresh tissue or large amounts of tissue adhering to
the specimen and/or no marrow inside the bone) should be extracted in Labs 9 and 10 if to
be used for mitochondrial DNA analysis.
2. Ensure that all reagents, instruments, and equipment satisfy the minimum standards for
quality control, where appropriate.
3. In all procedures,“label appropriately” means a minimum of AFDIL Case Number and

AFDIL Specimen Number.
4. Any laboratory workspace and all pipettors and racks to be used in this procedure must
be cleaned with 10% commercial bleach and thoroughly dried before beginning. When
using a laminar flow hood, expose to ultraviolet light for a minimum of 10 minutes after
cleaning.
5. Before use, laboratory instruments must be cleaned with 10% commercial bleach and
dried thoroughly (i.e. pipettes). In addition, they should be cleaned in the same manner
following the processing of each individual specimen.
Page 22 of 70
6. Microcentrifuge tubes (closed) should be irradiated in an ultraviolet crosslinker with 2
J/cm2. Extraction buffer and TE buffer (in 50 ml conical tubes), if used, must be
irradiated with 6 J/cm2. Follow "Irradiation of Reagents and Supplies in the Ultraviolet
Crosslinker" SOP for appropriate method of irradiation.
7. No reagents used for the extraction of DNA from specimens will be allowed in a post-
amplification room.
8. Any supplies or equipment taken from a post-amplification room to a pre-amplification

room must be sterilized with 10% commercial bleach before removal from the post-
amplification room and again in the pre-amplification room before use.
9. Only one evidence specimen should be open at any one time. For CILHI cases, only
evidence specimens from one case should be extracted together. For OAFME cases and
databasing, more than one case can be extracted at a time.
10. A minimum of one reagent blank should be carried throughout the extraction procedure
and assayed in parallel with the evidence samples. If appropriate, a substrate control
should be carried throughout the extraction and assayed in parallel with the evidence.
Order of processing for each procedure is as follows: substrate control(s), specimen(s),
reagent blank(s).
11. Change pipet tips between each transfer or addition of sample or reagent, unless
otherwise noted.
12. No aliquot of any reagent may be returned to the original stock container.
13. If more than four specimens are processed at any one time for mtDNA ID cases, an
additional reagent blank must be initiated (one RB for every 4 specimens). For OAFME
and mass disaster cases, more than four specimens from the same case can be processed
at a time.
14. For mitochondrial cases only: When a sufficient amount of sample is remaining, a
second extraction, performed at a different time (e.g., the day following completion of the
first extraction), will be conducted on each evidentiary specimen.
15. Use aerosol-resistant pipet tips for all liquid transfers.
V. SAFETY
1. All appropriate MSDS sheets must be read prior to performing this procedure.
2. All organic extraction procedures are to be performed in a chemical fume hood.
3. Treat all biological specimens as potentially infectious. Gloves, safety glasses, and a
laboratory coat must be worn at all times.
4. When performing the organic extraction steps, either VitonTM or 6 mil or greater nitrile
gloves must be worn. If using nitrile gloves, each hand must be double-gloved. The
outer glove on each hand must be removed immediately if an organic solvent is observed
on the glove. If a significant amount of solvent is observed, both gloves on that hand
Page 23 of 70
must be replaced. Inspect gloves approximately every five minutes for wetness or spots
from solvents.
5. Follow the "Exposure Control Plan for Occupational Exposure to Bloodborne Pathogens"
(DoD Forensic Advisory Committee).
6. Avoid direct exposure to ultraviolet light when using the germicidal lamp in the
biological hood or the transilluminator.
7. Distinguish all waste as general, biohazard, organic, chemical or SharpsTM and discard
appropriately.
Page 24 of 70
VI. PROCEDURES & WORKSHEETS – ORGANIC EXTRACTIONS
Page 25 of 70
A. Organic Extraction of DNA from Whole Blood – Procedure
Note: Liquid blood should be mixed well before removing aliquots for extraction or
storage. Blood should be stored at 4°C for five days at most before being
aliquotted and frozen. Blood can be frozen in approximately 1 ml aliquots.
1. Thaw blood and transfer 700 µl to a 1.7 ml tube. Add 800 µl of 1X SSC and mix. Spin 1
minute in microfuge.
2. Remove and discard 1.0 ml supernatant into disinfectant.
3. Add 1.0 ml 1X SSC, vortex, centrifuge 1 minute, remove all supernatant fluid. Do not
remove pellet.
4. To the pellet add: 375 µL 0.2M Sodium Acetate

25 µL 10% SDS
5 µL Proteinase K (20 mg/ml H20)
5. Vortex briefly (1 second) and incubate at 56˚C for 1 hour.
6. Add 120 µL phenol/chloroform/isoamyl alcohol; vortex 30 sec. This step must be carried
out in the fume hood.
7. Spin for 2 minutes.
8. The aqueous layer is carefully removed and placed in a new 1.7 ml tube. Do not remove
the layer of denatured protein that collects at the interface. The new tube now contains the
DNA; the old tube containing the phenol and denatured protein can be discarded.
9. To the aqueous layer add 1.0 ml cold, absolute EtOH. Mix by inversion of the tube. Place
the tube at -20˚C for a minimum of 15 minutes. Spin for 2 minutes.
10. Remove and discard the supernatant fluid by decantation. Use a pipette to remove any
additional alcohol. Be careful to avoid drawing any of the pellet up into the tip.
11. To the pellet add 180 µL TE. Vortex. Incubate at 56˚C for 10 minutes. Add 20 µl 2.0M
Sodium Acetate and mix 5 seconds by hand.
12. Add 500 µL cold, absolute EtOH. Mix gently by hand to achieve a homogeneous solution.
Spin for 1 minute. Remove the supernatant by decantation.
13. Wash the pellet with 1.0 ml room temperature 70% EtOH. Spin 1 minute and decant
supernatant fluid. Remove the maximum quantity of EtOH with a micropipette.
Page 26 of 70
14. Place tubes in the Hetovac centrifuge to remove excess EtOH. This process should take
about 10 minutes.
15. Add 200 µL TE-4, mix, and incubate at 56˚C overnight. Vortex following the incubation
and briefly spin (1 sec).
17. Store at 4˚C if amplified within 3 weeks or used routinely. Store at -20˚C for long-term
storage but limit the number of freeze/thaw cycles.
Note: Final volume of extract depends upon the level of DNA presumed to be present
based on visual observation of the evidence and/or specific case history.
Organic Extraction of DNA from Whole Blood - DNA Form 211
Page 27 of 70
Organic Extraction of DNA from Whole Blood
Specimen Comments
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Extraction Components
Item Source Lot #
Extraction Buffer AFDIL QC
Proteinase K AFDIL QC
Phenol/Chloroform/Isoamyl Alcohol AFDIL QC
1-Butanol AFDIL QC
TE Buffer AFDIL QC
®
Centricon-100 Amicon
1. Thaw blood and transfer 700 µl to a 1.7 ml tube. Add 800 µl of 1X SSC and mix. Spin 1 minute in microfuge.
2. Remove and discard 1.0 ml supernatant into disinfectant.
3. Add 1.0 ml 1X SSC, vortex, centrifuge 1 minute, remove all supernatant fluid. Do not remove pellet.
4. To the pellet add: 375 µL 0.2M Sodium Acetate, 25 µL 10% SDS, and 5 µL Proteinase K (20 mg/ml)
5. Vortex briefly (1 second) and incubate at 56˚C for 1 hour.
6. Add 120 µL phenol/chloroform/isoamyl alcohol; vortex 30 sec. This step must be carried out in the fume hood.
7. Spin for 2 minutes.
8. The aqueous layer is carefully removed and placed in a new 1.7 ml tube. Do not remove the layer of denatured protein that collects at the
interface. The new tube now contains the DNA; the old tube containing the phenol and denatured protein can be discarded.
9. To the aqueous layer add 1.0 ml cold, absolute EtOH. Mix by inversion of the tube. Place the tube at -20˚C for a minimum of 15 minutes. Spin
for 2 minutes.
10. Remove and discard the supernatant fluid by decantation. Use a pipette to remove any additional alcohol. Be careful to avoid drawing any of
the pellet up into the tip.
11. To the pellet add 180 µL TE. Vortex. Incubate at 56˚C for 10 minutes. Add 20 µl 2.0M Sodium Acetate and mix 5 seconds by hand.
12. Add 500 µL cold, absolute EtOH. Mix gently by hand to achieve a homogeneous solution. Spin for 1 minute. Remove the supernatant by
decantation.
13. Wash the pellet with 1.0 ml room temperature 70% EtOH. Spin 1 minute and decant supernatant fluid. Remove the maximum quantity of
EtOH with a micropipette.
14. Place tubes in the Hetovac centrifuge to remove excess EtOH. This process should take about 10 minutes.
15. Add 200 µL TE-4, mix, and incubate at 56˚C overnight. Vortex following the incubation and briefly spin (1 sec).
17. Store at 4˚C if amplified within 3 weeks or used routinely. Store at -20˚C for long-term storage but limit the number of freeze/thaw cycles.
Comments: ____________________________________________________________________________________
Location of DNA Extracts ____________________________________________________________________________
DNA Form 211 030101
Page 28 of 70
B. Organic Extraction of DNA from Bloodstains – Procedure
Special Supply: Spin-EASE™ Tubes. If no Spin-EASE tubes are available, a 0.5 ml thin-wall
tube with a hole pierced into the bottom inside a 1.7 ml tube can be substituted.
0.5 ml tube with hole = basket portion of Spin-EASE cartridge
1.7 ml tube = lower portion of Spin-EASE cartridge
1. Label all tubes with case number and specimen number. Cut a part of the stain
(approximately 3 mm2, or larger if necessary) into small pieces and place into the lower
portion of a sterile, labeled Spin-EASE extraction tube.
Note: If available, a similar-sized cutting of an unstained portion of the substrate should

be prepared to act as a substrate control. This should be performed before any
evidentiary stains are cut.
2. Add 400 µl extraction buffer (ensure that the extraction buffer is homogenous) and 10 µl
20 mg/ml proteinase K to each specimen and the controls. Mix thoroughly and pulse
spin.
3. Incubate at 56oC overnight.
4. Perform piggy-back spin as follows: Transfer the stain cutting to the sterile, labeled
basket portion of a Spin-EASE extraction tube. Place the basket into the lower portion of
the Spin-EASE tube. Spin for 2 minutes at 10,000-15,000 rpm in a microcentrifuge.
5. Place the Spin-EASE basket aside to allow the stain cutting to air dry overnight. When
dried, repackage and store as evidence.
6. Add 400 µl phenol/chloroform/isoamyl alcohol (25:24:1) to extract volume from Step 4.

Mix thoroughly. Spin for 2 minutes at 10,000-15,000 rpm in a microcentrifuge. Transfer
upper aqueous layer to a sterile, labeled microcentrifuge tube. Repeat extraction with
phenol/chloroform/isoamyl alcohol until the interface is clean, or a minimum of two
times. Dispose of phenol waste in the appropriate waste container.
7. Add 400 µl n-Butanol to extract volume. Mix thoroughly. Spin for 2 minutes at 10,000
– 15,000 rpm in a microcentrifuge. Remove and discard most of the butanol upper layer
into the appropriate waste container.
Perform in Laminar Flow Hood
8. Assemble a sufficient number of Centricon® -100 concentrators and label appropriately.

Add 1 ml of sterile TE Buffer to sample reservoir. Recap and transfer to chemical fume
hood.
Page 29 of 70
Perform in Chemical Fume Hood
9. Transfer the lower aqueous layer of each sample to its corresponding Centricon
concentrator. Try to avoid pipetting any residual n-Butanol. Centrifuge approximately 30
minutes, or longer if necessary, at 1000 x g (2600 RPM) using the IEC Centra MP4.
Discard filtrate.
10. Add 2 ml TE buffer to each of the Centricon concentrators and spin approximately 30
Repeat.
11. Pipette extract directly from the sample reservoir and transfer to a sterile labeled
microcentrifuge tube. Add this liquid to the labeled microcentrifuge tube and resuspend
extract to a final volume of approximately 100 µl.
Note: Take extreme care in not allowing the pipettor to touch the sides of the sample
reservoir.
13. Store samples at 4°C if to be amplified within 3 weeks or used routinely. Store at
-20°C for long-term storage, but limit the number of freeze/thaw cycles.
Organic Extraction of DNA from Bloodstains - DNA Form 178
Page 30 of 70
Organic Extraction of DNA from Bloodstains
Extraction
Item Source Lot #
1-Butanol AFDIL QC
TE Buffer AFDIL QC
Centricon-100® Amicon
Centricon-100® Purification
Approximate Approximate
# Specimen Volume Recovered (in µl) Final Volume (in µl) Comments
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Reagent Blank #:
1. Add 400 µl extraction buffer to stain cutting in Spin-EASE™ tube or microcentrifuge tube.
2. Add 10 µl of 20 mg/ml proteinase K.
4. Transfer stain cutting to the basket portion of the Spin-EASE tube. Replace basket in lower portion of the Spin-
EASE tube. Spin for 2 minutes at 10,000-15,000 rpm in a microcentrifuge.
5. Add 400 µl phenol/chloroform/isoamyl alcohol to extract volume. Mix thoroughly.
6. Centrifuge 2 minutes at 10,000-15,000 rpm in a microcentrifuge.
7. Transfer upper aqueous layer to a clean microcentrifuge tube.
8. Repeat steps 5, 6, and 7 until interface is clean.
9. Add 400 µl n-butanol. Mix thoroughly. Centrifuge 2 minutes at 10,000-15,000 rpm in a microcentrifuge.
Remove and discard most of the butanol upper layer into the appropriate waste container.
10. Label a sufficient number of pre-assembled Centricon®-100 concentrators.
11. Add 1 ml TE buffer to the sample reservoir of the Centricon-100. Recap before transfer to the chemical fume
hood.
12. Transfer the lower aqueous layer to the sample reservoir of the Centricon-100. Avoid pipetting any residual
butanol. Spin column at 1000 x g (IEC 2600 rpm) for 15-30 minutes or until sample has spun through.
13. Add 2 ml TE buffer and spin 15-30 minutes at 1000 x g or until the buffer has spun through. Discard filtrate and
repeat 2 ml TE buffer wash.
14. Transfer the retentate to a sterile, labeled microcentrifuge tube. Adjust final volume to approximately 100 µl with
TE buffer.
Comments: ___________________________________________________________________________________
Location of DNA Extracts ________________________________________________________________________
Page 31 of 70
C. Organic Extraction of DNA from Tissue – Procedure
1. Cut approximately a 3 - 5 mm3 section of tissue into small pieces and place into a 1.7 ml
tube labeled with case number and specimen number. If the sample is highly degraded
larger portions of tissue may be extracted in 15 ml conical tubes. Adjust all reagent
volumes accordingly.
2. Add 500 µl extraction buffer and 10 µl proteinase K (20 mg/ml). (assure that the Extraction
Buffer is homogenous and prewarmed to 56˚C). Mix thoroughly and pulse spin.
4. Add 500 µl phenol/chloroform/isoamyl alcohol. Vortex for 30 seconds.
5. Centrifuge at 10,000-15,000 rpm for 2 minutes.
6. Transfer upper aqueous layer to a clean, labeled tube. Repeat extraction with
phenol/chloroform/isoamyl alcohol until the interface is clean, or a minimum of two times.
Dispose of phenol waste in the appropriate waste container. The new tube now contains
the DNA; the old tube containing the phenol and denatured protein can be discarded.
7. Extract with 500 µl n-Butanol. Mix well and centrifuge 2 minutes at 10,000 - 15,000 rpm
in a microcentrifuge.
8. Assemble a sufficient number of Centricon® -100 concentrators and label appropriately.

Add 1 ml of sterile TE Buffer to sample reservoir. Recap and transfer to chemical fume
hood.
9. Transfer the lower aqueous layer of each sample to its corresponding Centricon
concentrator. Try to avoid pipetting any residual n-Butanol. Centrifuge approximately 30
Discard filtrate.
10. Add 2 ml of sterile TE Buffer to a sample reservoir. Centrifuge for approximately 15

minutes or longer if necessary at 1000 x g (IEC 2600 RPM). Discard filtrate. Repeat.
microcentrifuge tube. Resuspend extract to a final volume of approximately 50 µl.
reservoir.
Page 32 of 70
13. Store samples at 4˚C if to be amplified within 3 weeks or used routinely. Store at
-20˚C for long-term storage, but limit the number of freeze/thaw cycles.
"Organic Extraction of DNA from Soft Tissue"/DNA Form 154
Page 33 of 70
Organic Extraction of DNA from Tissue

Extraction
Item Source Lot #
1-Butanol AFDIL QC
TE Buffer AFDIL QC
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Reagent Blank #:
1. Add approximately 3-5 mm3 piece to a labeled 1.7 ml tube.
2. Add 500 µl extraction buffer and 10 µl 20 mg/ml proteinase K. Mix and spin 2 seconds at 10,000-15,000 rpm.
3. Incubate at 56°C overnight.
4. Add 500 µl phenol/chloroform/isoamyl alcohol. Vortex.
5. Spin 2 minutes at 10,000-15,000 rpm.
6. Transfer upper aqueous layer to a sterile 1.7 ml tube.
7. Repeat steps 4, 5 and 6 until interface is clean.
8. Add 500 µl n-butanol. Mix thoroughly. Spin 2 minutes at 10,000-15,000 rpm.
9. Assemble and label a sufficient number of Centricon 100 concentrators.
10. Add 1 ml TE buffer to the sample reservoir of the Centricon 100. Recap and transfer to the chemical fume hood.
11. Transfer the lower aqueous layer to the sample reservoir.
12. Spin column at 1000 x g (IEC 2600 rpm) for 15-30 minutes or until sample has spun through.
13. Add 2 ml TE buffer and spin at 1000 x g (IEC 2600 rpm) for 15-30 minutes or until sample has spun through.
Repeat.
14. Transfer retentate to a sterile, labelled tube. Adjust final volume to approximately 50 µl.
Comments:________________________________________________________________________________
__________________________________________________________________________________________
DNA Form 154 012601
Page 34 of 70
D. Organic Extraction of DNA from Fresh Bone – Procedure
Special Equipment: Waring Blender Unit for grinding bone into powder; Mortar, chisel and
hammer.
1. Obtain a bone specimen and record its gross weight.
2. Cover the specimen in a 50 ml conical tube with 95% ethanol. Shake the tube at least 10
times. Decant the ethanol into a waste container. Repeat twice. Pour the bone fragments
into a clean labeled weigh boat and allow to air dry in the laminar flow hood.
3. Crush the bone fragment into smaller pieces using a clean mortar, chisel and hammer. If
the bone specimen is dry proceed with step 4. However, if marrow is still present inside
the bone, empty the contents of the mortar into a 15 ml conical tube, labeled appropriately,
and skip to step 8.
4. Clean the grinding chamber of a Waring Blender and the lid: fill pulverizer ~ 1/3 full with
10% bleach, attach lid and run unit approximately 10 seconds; remove lid without touching
inside of lip, rinse lid and cup with sterile distilled water, then 95% ethanol. Drain off the
excess ethanol. Allow the interior surfaces to dry thoroughly.
5. Place specimen fragments into the lower portion of the grinding chamber. Place lid on
blender without touching inside lip of lid or cup.
6. Attach sealed blender cup to base unit. Run blender for up to one minute. If sample is not
completely ground after one minute, shut off blender, tap the container, and wait
approximately 30 seconds. Repeat if necessary. When starting the unit, the bone may
become wedged between a blade and the cup wall; if so, shut off, remove cup and dislodge
bone by tapping or rotating the blade spindle from below.
7. Tilt grinding chamber at approximately a 45 degree angle and tap gently on bench top to
accumulate the specimen to one side. Wait approximately 30 seconds for bone powder to
settle then gently remove lid. Pour powder into a clean weigh boat or funnel for transfer to
a labeled sterile, preweighed or tared 15 ml conical tube.
8. Determine the weight of the pulverized bone specimen in the conical tube.
9. Add 3 ml of extraction buffer (prewarmed to 56°C) and 100 µl 20 mg/ml proteinase K.
10. Incubate overnight at 56ºC.
11. Extract with 3 ml of Phenol/Chloroform/Isoamyl alcohol (25:24:1). Mix well and

centrifuge 2 minutes at 4950 X g using the Beckman GPR Centrifuge (6400 RPM) or the
IEC Centra MP4 (5000 RPM). Transfer upper aqueous layer to clean labeled tube. Repeat
extraction with Phenol/Chloroform/Isoamyl Alcohol a minimum of two times or until the
interface is clean. Dispose of phenol waste in the appropriate waste container.
12. Extract with 3 ml n-Butanol. Mix well and centrifuge 2 minutes at 4950 X g using the
Beckman GPR Centrifuge (6400 RPM) or the IEC Centra MP4 (5000 RPM).
Page 35 of 70
13. Assemble a sufficient number of Centricon-100® concentrators in the laminar flow hood.
Label appropriately.
14. Transfer the lower aqueous layer to a Centricon-100. Avoid pippetting any residual n-
Butanol. Centrifuge for approximately 15 minutes, or longer if necessary at 1000 x g (IEC
2600 RPM). Discard filtrate.
15. Add 2 ml of sterile TE Buffer to the sample reservoir. Centrifuge for approximately 15
microcentrifuge tube. Add this liquid to the labeled microcentrifuge tube and resuspend extract
to a final volume of approximately 100 µl (or adjust the final volume of extract dependant upon
the level of DNA presumed to be present based on visual observation of the evidence and/or
specific case history).
reservoir.
18. Store at 4°C if amplified within 3 weeks or used routinely. Store at -20°C for long-term
"Organic Extraction of Fresh Bone"/DNA Form 210
Page 36 of 70
Organic Extraction of DNA from Fresh Bone
Followed Version _____ of the "AFDIL Extraction Manual"
Case Number:_________________________Date:____________________Scientist:_____________________________
Extraction
Item Source Lot #
1-Butanol AFDIL QC
TE Buffer AFDIL QC
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Reagent Blank #:
1. Add 3 ml extraction buffer to pulverized specimen in 15 ml conical tube.

4. Add 3 ml phenol/chloroform/isoamyl alcohol (25:24:1). Mix thoroughly.
5. Centrifuge 2 minutes at 4950 x g (Beckman 6400 rpm/IEC 5000rpm).
6. Transfer upper aqueous layer to a sterile, labelled 15 ml conical tube.
7. Perform steps 4, 5, and 6 a minimum of two times or until the interface is clean.
8. Add 3 ml n-butanol. Mix thoroughly.
9. Centrifuge 2 minutes at 4590 x g.
10. Add lower aqueous layer from Butanol extract to sample reservoir of Centricon-100.
11. Spin column at 1000 x g (IEC 2500 RPM) for approximately 15 minutes or until sample has spun through.
12. Add 2 ml TE Buffer and spin for approximately 15 minutes or until TE Buffer has spun through. Discard filtrate
and repeat 2 ml TE Buffer wash.
13. Transfer retentate to a sterile, labelled tube. Adjust final volume to approximately 100 µl with TE.
Comments: ____________________________________________________________________________________
___________________________________________________________________________________________________
Location of DNA Extracts _________________________

Page 37 of 70
E. Organic Extraction of DNA from Swabs – Procedure
Special Supplies: Spin-EASE™ Tubes. If no Spin-EASE tubes are available, a 0.5 ml thin-wall
tube with a hole pierced into the bottom inside a 1.7 ml tube can be substituted.
1. Cut half the swab (or whole swab if necessary) off the applicator stick, tease apart with
forceps, and place into a 1.7 ml tube.
2. Add 800 µl extraction buffer (prewarmed to 56ºC) and 20 µL proteinase K (20 mg/ml).
3. Incubate at 560C overnight.
4. Pulse vortex 10 - 15 seconds.
5. Spin for 15 minutes at 10,000 - 15,000 rpm. Remove and discard 0.5 ml of supernatant.
6. Press excess liquid out of the swab and perform a piggy back spin as follows: Transfer the
swab to the sterile, basket portion of a Spin-EASE extraction tube. Place the basket into
the lower portion of the Spin-EASE tube. Spin for 1 minute at 10,000 - 15,000 rpm.
7. Remove swab from basket and place into a weigh boat to air dry. Once swab is dry,
repackage and store at -20˚C.
8. Add 800 µL Phenol/Chloroform/Isoamyl Alcohol(25:24:1); vortex 30 sec.
9. Spin at 10,000 - 15,000 for 2 minutes.
10. Transfer upper aqueous layer to a clean labeled tube. Repeat extraction with
Phenol/Chloroform/Isoamyl Alcohol until the interface is clean, or a minimum of two
times. Dispose of phenol waste in the appropriate waste container. The new tube now
contains the DNA; the old tube containing the phenol and denatured protein can be
discarded.
11. Extract with 800 µl n-Butanol. Mix well and spin 2 minutes at 10,000 - 15,000 rpm in a
microcentrifuge.
12. Add 1.0 ml TE buffer to the sample reservoir of the Centricon-100. Recap before transfer
to the chemical fume hood.
Page 38 of 70
13. Transfer the lower aqueous layer to a Centricon-100. Avoid pipetting any residual n-
Butanol. Spin for approximately 15 minutes or longer, if necessary, at 1000 x g (IEC 2600
RPM). Discard filtrate.
14. Add 2 ml of sterile TE Buffer to a sample reservoir. Spin for approximately 15 minutes or
longer if necessary at 1000 x g (IEC 2600 RPM). Discard filtrate. Repeat.
microcentrifuge tube. Resuspend extract with TE to a final volume of approximately 100
µl (or adjust the final volume of extract dependant upon the level of DNA presumed to be
present based on visual observation of the evidence and/or specific case history).
reservoir.
16. Store at 4°C if amplified within 3 weeks or used routinely. Store at -20°C for long term
“Organic Extraction of DNA from Swabs” - DNA Form 45
Page 39 of 70
Organic Extraction of DNA from Swabs
Followed Version _____ of the "AFDIL DNA Extraction Manual"
Case Number:_________________________Date:____________________Scientist:_____________________________
Extraction
Item Source Lot #
1-Butanol AFDIL QC
TE Buffer AFDIL QC
®
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Reagent Blank #:
1. Cut the swab off the applicator stick, tease, and place into a 1.7 ml tube.
2. Add 800 µl extraction buffer and 20 µL proteinase K (20 mg/ml).
3. Incubate at 560C overnight.
4. Vortex 10 - 15 seconds.
5. Spin for 15 minutes at 10,000 - 15,000 rpm. Remove and discard 0.5 ml of supernatant.
6. Press excess liquid out of the swab and perform a piggy back spin as follows: Transfer the swab to the sterile, basket portion of a
Spin-EASETM extraction tube. Place the basket into the lower portion of the Spin-EASE tube. Spin for 3 minutes at 10,000 -
15,000 rpm.
7. Remove swab from basket, package and store it as evidence. Discard basket.
8. Add 800 µL phenol/chloroform/isoamyl alcohol(25:24:1); vortex 30 sec.
9. Spin at 10,000 - 15,000 for 2 minutes.
10. Transfer upper aqueous layer to a clean labelled tube. Repeat extraction until the interface is clean, or a minimum of two times.
11. Add 800 µl n-Butanol. Mix well and spin 2 minutes at 10,000 - 15,000 rpm in a microcentrifuge.
12. Label a sufficient number of Centricons and transfer to chemical fume hood.
13. Transfer the lower aqueous layer to a Centricon-100. Spin for approximately 15 minutes, or longer if necessary at 1000 x g (IEC
14. Add 2 ml of sterile TE Buffer to a sample reservoir. Spin for approximately 15 minutes or longer if necessary at 1000 x g (IEC
2600 RPM). Discard filtrate. Repeat.
15. Pipette extract directly from the sample reservoir and transfer to a sterile labelled microcentrifuge tube. Resuspend the extract to
a total volume of 100µl.
Comments: ____________________________________________________________________________________
Page 40 of 70
F. Organic Extraction of DNA from Hair Roots – Procedure
Special Reagents, Supplies and Equipment: Cotton swabs (sterile, individually wrapped),
Dithiothreotol (DTT) - 1 M, Mounting media solvent (e.g., Toluene, Xylenes), Terg-a-zyme®
(5%), Centricon-30® concentrators, Glass microscope slides, Microscope, Sonicating water bath
1. If necessary, a qualified hair examiner will observe hair(s) microscopically.
2. Proceed to Step 3 if hairs are mounted; proceed to Step 12 if hairs are loose.
Mounted Hair(s)
Perform in the Chemical Fume Hood
CAUTION: Perform Steps 3 and 4 near the center of the hood to avoid losing sample in the
down draft of laminar flow at the front and back.
3. Place several drops of the appropriate mounting media solvent around the perimeter of the
coverslip.
Notes: 100% xylenes dissolves Permount; toluene dissolves Cytoseal. When using
mounting media solvents, 6 mil or greater nitrile gloves must be worn and each hand must
be double-gloved. The outer glove on each hand must be removed immediately if an
organic solvent is observed on the glove. If a significant amount of solvent is observed,
both gloves on that hand must be replaced. Inspect gloves approximately every five
minutes for wetness or spots from solvents. It may be necessary for sample to incubate at
room temperature for several minutes to allow mounting media to dissolve.
4. Carefully remove coverslip.
5. Using forceps, carefully remove hair(s) and place in a sterile, labeled (with case number
and specimen number) microcentrifuge tube containing 1.0 ml mounting media solvent.
Recap tube.
Note: Initiate reagent blank(s) at this step. Set up the evidentiary specimen(s) and then
the reagent blank(s).
6. Incubate at room temperature for approximately five (5) minutes. Gently agitate several
times during incubation.
7. Carefully remove with a pipette the mounting media solvent.
8. Add 1.0 ml sterile, distilled water. Gently agitate hair(s) to rinse off the mounting media
solvent.
9. Carefully remove the water with a pipette.
10. Repeat wash step one time and remove water.
11. Proceed to Step 12.
Page 41 of 70
Loose Hair(s)
12. Make a fresh 5% Terg-a-zyme® solution by adding 2.5 grams of Terg-a-zyme® to 50 ml of

deionized water. Incubate at 56 oC if necessary to allow for the Terg-a-zyme® to go into
solution.
13. Carefully transfer the hair with forceps to a weigh boat.
14. If it is apparent that there is biological material or other debris on the hair, swab the hair as
follows: Open a sterile swab. Wet the swab with deionized water and repeatedly swab the
hair (root and shaft) to remove any potential contamination. Label the swab with tape and
place the swab in a rack to dry. Once swab is dry, transfer the cotton swab head to a
sterile, labeled 1.7 ml microcentrifuge tube and store at 4 oC. Initiate a substrate control at
this step by collecting the swab head from an unused swab with the same lot number as
those used for swabbing the hair(s). Store at 4 oC. Testing of the swab will be determined
on a case by case basis.
15. Cut the hair close to the root end. Place the hair root portion into a sterile, labeled 1.7ml
microcentrifuge tube.
16. Add 1.0 ml of 5% Terg-a-zyme® solution.
Note: Initiate reagent blank(s) at this step if a loose hair was used. If a mounted hair
was used then a reagent blank should have been initiated in step 4. Set up the evidentiary
specimen(s) and then the reagent blank(s).
17. Place the tubes in a sonicating water bath for ten minutes.
18. Carefully remove with a pipette the 5% Terg-a-zyme® solution making sure not to lose the
hair root. Repeat Steps 15-17 once for a total of two washes.
19. Add 1.0 ml absolute ethanol. Recap tube and invert several times.
20. Carefully remove with a pipette the absolute ethanol making sure not to lose the hair root.
21. Add 1.0 ml deionized water. Recap tube and invert several times.
22. Carefully remove with a pipette the deionized water making sure not to lose the hair root.
23. Prepare hair lysis buffer (for 10 samples) by combining 1.87 ml of Extraction Buffer
(prewarmed to 56º), 80 µl of 1 M DTT, and 50 µl of 20 mg/ml Proteinase K.
Note: Hair lysis buffer must be prepared fresh daily.
24. Add 200 µl hair lysis buffer. Mix thoroughly.
25. Incubate at 56oC for a minimum of 2 hours.
Note: Make sure hair root is submerged and not sticking to the side of the tube.
26. Observe each sample for complete extraction such that no visible pieces of hair or tissue
remain. Proceed to Step 27 if all sample extracts appear homogeneous.
Page 42 of 70
27. If a sample extract does not appear homogeneous, add 5 µl 20 mg/ml proteinase K and 8 µl
1 M DTT and incubate at 56oC for 2-3 hours. Repeat as necessary (maximum of 5 times).
Note: Additional incubations with proteinase K and DTT must be performed with each
sample, including the reagent blank(s) and substrate control(s).
28. Add 200 µl phenol/chloroform/isoamyl alcohol (25:24:1). Mix thoroughly. Centrifuge for
2 minutes at 10,000-15,000 rpm in a microcentrifuge. Transfer upper aqueous layer to a
sterile, labeled microcentrifuge tube. Repeat phenol/chloroform/isoamyl alcohol extraction
until the interface is clean. Dispose of phenol waste in the appropriate waste container.
29. Add 200 µl n-butanol. Mix thoroughly. Centrifuge 2 minutes at 10,000-15,000 rpm in a
microcentrifuge. Remove and discard most of the n-butanol upper layer into the
appropriate waste container.
30. Label a sufficient number of pre-assembled Centricon-30® concentrators.
31. Add 1.5 ml TE buffer to the sample reservoir of the Centricon-30. Recap before transfer to
the chemical fume hood.
32. Transfer the lower aqueous layer to the sample reservoir of the Centricon-30. Avoid
pipetting any residual n-butanol. Centrifuge column at 1000 x g (IEC 2600 rpm) for 15-30
minutes or until sample has spun through. Discard filtrate.
33. Add 2 ml TE buffer and centrifuge 15-30 minutes at 1000 x g or until the buffer has spun
through.
34. Transfer retentate to a sterile, labeled microcentrifuge tube. Adjust final volume to
approximately 50 µl by adding the necessary amount of TE buffer.
Notes: Final volume of extract depends upon the level of DNA presumed to be present
based on visual observation of the evidence and/or specific case history. Take extreme
care in not allowing the pipettor to touch the sides of the sample reservoir.
36. Store samples at 4 oC if to be amplified within 3 weeks or used routinely. Store at

-20 oC for long-term storage, but limit the number of freeze/thaw cycles.

“Organic Extractionof DNA from Hair Roots” – DNA Form 34
Page 43 of 70
Organic Extraction of DNA from Hair Roots
Version _____ of the "AFDIL DNA Extraction Manual"
Item Source Lot #
Mounting Media Solvent (If necessary)
Sterile Distilled Water
Sterile Swabs
Terg-a-zyme® Alconox
Extraction Buffer AFDIL
1 M DTT AFDIL
Proteinase K AFDIL
Phenol:Chloroform:Isoamyl Alcohol AFDIL
1-Butanol AFDIL
TE Buffer AFDIL
# Specimen Size of Hair Extracted Approximate Volume Recovered Approximate Volume Recovered Comments
(Length in mm) (in µl) (in µl)
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Reagent Blank:
If Hair is mounted:
1. Place several drops of mounting media solvent around the perimeter of the cover slip.
2. Remove cover slip.
3. Place hair in microcentrifuge tube containing 1.0 ml of mounting media solvent.
4. Incubate at room temperature for 5 minutes. Gently agitate several times during incubation.
5. Carefully remove mounting media solvent.
6. Add 1.0 ml of sterile distilled water and gently agitate.
7. Carefully remove sterile distilled water.
8. Repeat water wash.
If Hair is loose:
9. Make a fresh 5% Terg-a-zyme® solution and incubate it at 56 oC (2.5 g Terg-a-zyme® in 50 ml sterile distilled water).
10. Transfer hair to a weigh boat and swab the hair repeatedly with a water soaked swab. Set the swab aside to dry.
11. Cut the hair between 5 and 10 mm in length. Transfer the hair root and remaining shaft to a microcentrifuge tube.
12. Add 1.0 ml of 5% Terg-a-zyme® solution.
13. Incubate in a sonicating water bath
®
for ten minutes.
14. Carefully remove Terg-a-zyme
®
solution.
15. Repeat Terg-a-zyme wash.
16. Add 1.0 ml 100% ethanol. Recap and invert several times.
17. Carefully remove 100% ethanol.
18. Add 1.0 ml sterile distilled water. Recap and invert several times.
19. Carefully remove sterile distilled water.
20. Prepare fresh hair lysis buffer (187 µl of Extraction Buffer, 8 µl of 1M DTT, and 5 µl of 20 mg/ml Proteinase K per sample).
21. Add 200 µl hair lysis buffer to hair(s) in microcentrifuge tube.
22. Incubate at 56oC for a minimum of 2 hours. If hair does not dissolve add an additional 8 µl DTT and 5 µl Proteinase K to
each sample that is not homogeneous and to the reagent blank.
23. Incubate at 56oC for 2 hours. Repeat as necessary (maximum of 5 times)
24. Add 200 µl Phenol/Chloroform/Isoamyl Alcohol.
25. Centrifuge 2 minutes at 10,000-15,000 rpm in a microcentrifuge.
26. Transfer upper aqueous layer to a clean microcentrifuge tube.
28. Add 200 µl n-butanol. Mix thoroughly. Centrifuge 2 minutes at 10,000-15,000 rpm in a microcentrifuge. Remove and
discard most of the n-butanol upper layer into the appropriate waste container.
29. Label a sufficient number of pre-assembled Centricon-30® concentrators.
30. Add 1.5 ml TE buffer to the sample reservoir of the Centricon-30®. Recap before transfer to the chemical fume hood.
31. Transfer the lower aqueous layer to the sample reservoir of the Centricon-30®. Avoid pipetting any residual n-butanol. Spin
column at 1000 x g (IEC 2600 rpm) for 15-30 minutes or until sample has spun through. Discard filtrate.
32. Add 2 ml TE buffer and spin 15-30 minutes at 1000 x g or until the buffer has spun through. Discard filtrate.
33. Transfer retentate to a sterile, labeled microcentrifuge tube.
34. Adjust final volume to approximately 50 µl.
Comments: ____________________________________________________________________________________________
DNA Form 34 revised 022601
Page 44 of 70
G. Organic Extraction of DNA from Hair Shafts – Procedure
Special Reagents, Supplies and Equipment: Cotton swabs (sterile, individually wrapped),
Dithiothreotol (DTT) - 1 M, Mounting media solvent (e.g., Toluene, Xylenes), Terg-a-zyme®
(5%), Centricon-30® concentrators, Glass microscope slides, Microscope, Sonicating water bath,
Micro tissue Grinder
Begin at Step 1 if hairs are mounted. If hairs are loose, begin at Step 11.
Notes: A "specimen" refers to a single hair shaft or pool of up to approximately 15 hairs.
At the examiner's discretion, it is allowable to extract only a portion of a single hair

specimen.
If necessary, a "bulk extraction" may be performed. Adjust extraction volumes and

sample containers accordingly (See steps 37-49).
If microscopic analysis is necessary, it must be performed by a qualified hair examiner.
In all cases where Extraction Buffer is used, be sure it is prewarmed to 56ºC and is
homogeneous.
Do not clean and reuse micro tissue grinders. Use a fresh one for each extraction.
Mounted Hair(s) – Use “Organic Extraction of DNA from Mounted Hairs” – DNA Form 37 for
this procedure.

2. Place several drops of the appropriate mounting media solvent around the perimeter of
the coverslip.
Notes: 100% xylenes dissolves Permount; toluene dissolves Cytoseal. When using
mounting media solvents, 6 mil or greater nitrile gloves must be worn and each
hand must be double-gloved. The outer glove on each hand must be removed
immediately if an organic solvent is observed on the glove. If a significant
amount of solvent is observed, both gloves on that hand must be replaced. Inspect
gloves approximately every five minutes for wetness or spots from solvents.
It may be necessary for the sample to incubate at room temperature for several
minutes to allow mounting media to dissolve.
CAUTION: Perform Steps 3 and 4 near the center of the hood to avoid losing sample in the
down draft of laminar flow at the front and back.
3. Carefully remove coverslip.
4. Using forceps, carefully remove hair(s) and place in a sterile, labeled microcentrifuge
tube containing 1.0 ml mounting media solvent. Recap tube.
5. Incubate at room temperature for approximately five (5) minutes. Gently agitate several
times during incubation.
Page 45 of 70
6. Carefully remove the mounting media solvent.
7. Add 1.0 ml sterile, deionized water. Gently agitate hair(s) to rinse off the mounting
media solvent.
8. Carefully remove water.
10. Proceed to Step 12.
Loose Hair(s) – Use “Organic Extraction of DNA from Loose Hairs” – DNA Form 39 for this
procedure.
Perform in the General Laboratory


12. Clean micro tissue grinders with 10% bleach, water, and ethanol, in that order. Allow to
dry completely before using.
13. Irradiate the micro tissue grinders in the ultraviolet crosslinker with a minimum of 6
J/cm2 or the same amount of energy used for irradiating conical tubes as per the
"Irradiation of Reagents and Supplies in the Ultraviolet Crosslinker" protocol.
14 Carefully remove hair(s) and place in a different sterile, labeled, 1.7 ml microcentrifuge
tube.
15. Add 1.0 ml of 5% Terg-a-zyme solution. (2.5 g Terg-a-zyme® in 50 ml sterile distilled

water).
16. Place the tube in the sonicator for approximately twenty (20) minutes.
17. Remove the 1.7 ml tube from the sonicator and carefully remove the 5% Terg-a-zyme
solution.
18. Repeat steps 15-17 at least three times for a total of at least four washes or as needed. If
a hair examination or visual inspection indicates the presence of blood, debris, etc., then
use a new tube for each wash.
19. Rinse the hair with 1.0 ml of 100% ethanol. Recap tube. Gently agitate several times.
20. Remove the 100% ethanol. Rinse the hair with 1.0 ml of deionized H2O. Recap tube.
Gently agitate several times.
21. Carefully remove the H2O.
If extracting a large number of hairs so that a 15ml conical tube is necessary to

perform the extraction, go to step 37.
Page 46 of 70
22. Prepare reagent blank for each sample.
a) Add 187µl of Extraction buffer to micro tissue grinder.
b) Briefly simulate grinding.
c) Transfer the Extraction buffer to a labeled 1.7 ml microcentifuge tube.
23. To the same micro tissue grinder add 187µl of extraction buffer.
24. Place hair(s) in the micro tissue grinder.
25. Grind until fragments of hair are no longer visible.
26. Transfer the solution into a labeled 1.7 ml microcentrifuge tube.
27. Repeat steps 22-26 for all hairs.
28. Add 5µl of 20 mg/ml Proteinase K and 8µl of 1 M DTT to each reagent blank and sample
tube.
29. Incubate at 56 °C for a minimum of 2 hours.

30. Add 200 µl phenol/chloroform/isoamyl alcohol (25:24:1). Mix thoroughly. Spin for 2
minutes at 10,000-15,000 rpm in a microcentrifuge. Transfer upper aqueous layer to a
sterile, labeled microcentrifuge tube. If necessary, repeat extraction with
phenol/chloroform/isoamyl alcohol until the interface is clean. Dispose of phenol waste
in the appropriate waste container.

31. Add 200 µl n-butanol. Mix thoroughly. Centrifuge 2 minutes at 10,000-15,000 rpm in a

32. Label a sufficient number of pre-assembled Centricon-30®concentrators.
33. Add 1.5 ml TE buffer to the sample reservoir of the Centricon-30® . Recap before transfer
to the chemical fume hood.

34. Transfer the lower aqueous layer to the sample reservoir of the Centricon-30® . Avoid
pipetting any residual n-butanol. Spin column at 4000 x g (IEC 4500 rpm) for 15-30
35. Add 2 ml TE buffer and spin 15-30 minutes at 4000 x g using Centricon-30® or until the
buffer has spun through.
36. Go to Step 51
Large Volume Extractions
37. Prepare reagent blank for each sample.

Page 47 of 70
a.) Add 200µl of Extraction buffer to micro tissue grinder.
b.) Briefly simulate grinding.
c.) Transfer the Extraction buffer to a labeled 15 ml conical tube.
d.) Add 2.8 ml Extraction buffer to conical tube.
38. To the same micro tissue grinder add 200 µl of extraction buffer.
39. Place hair(s) in the micro tissue grinder.
41. Add additional hairs.
42. Transfer the solution into a labeled 15 ml microcentrifuge tube.
43. Repeat steps 37-41 for all specimens.
44. Add 100 µl of Proteinase K and 160 µl of DTT to each reagent blank and sample tube.
45. Incubate at 56 °C for 2-24 hours.

46. Add 3 ml phenol/chloroform/isoamyl alcohol (25:24:1). Mix thoroughly. Spin for 2
minutes at 10,000-15,000 rpm in a microcentrifuge. Transfer upper aqueous layer to a
sterile, labeled microcentrifuge tube. If necessary, repeat extraction with
phenol/chloroform/isoamyl alcohol until the interface is clean. Dispose of phenol waste
in the appropriate waste container.

47. Add 3 ml n-butanol. Mix thoroughly. Centrifuge 2 minutes at 10,000-15,000 rpm in a


49. Transfer the lower aqueous layer to the sample reservoir of the Centricon-30® . Avoid

50. Add 2 ml TE buffer and spin 15-30 minutes at 4000 x g using Centricon-30® or until the
buffer has spun through. Repeat once.
Page 48 of 70
reservoir.
53. Store samples at 4ºC if to be amplified within 3 weeks or used routinely. Store at
-20ºC for long-term storage, but limit the number of freeze/thaw cycles.
You will need the following worksheet(s) for this procedure:
“Organic Extraction of DNA from Loose Hairs” – DNA Form 39

“Organic Extraction of DNA from Mounted Hairs” – DNA Form 37
Page 49 of 70
Organic Extraction of DNA from Loose Hairs
Item Source Lot #

1 M DTT AFDIL
Proteinase K AFDIL
Phenol:Chloroform:Isoamyl Alcohol AFDIL
n-Butanol AFDIL
TE Buffer AFDIL
Mounting Media Solvent
# Specimen Amount Extracted Approximate Volume Recovered(µl) Approximate Final Volume(µl)
Specimen #:
Specimen#:
Specimen #:
Specimen #:
Reagent Blank #:
Reagent Blank #:
Reagent Blank #:
Reagent Blank #:
1. Clean micro tissue grinders with 10% bleach, water, and ethanol, in that order. Allow to dry completely before using.
2. Irradiate the micro tissue grinders in the ultraviolet crosslinker according to protocol.
3. Carefully remove hair(s) and place in a different sterile, labeled, 1.7 ml microcentrifuge tube.
4. Add 1.0 ml of 5% Terg-a-zyme solution and place the tube in the sonicator for approximately twenty (20) minutes.
5. Remove the 1.7 ml tube from the sonicator and carefully remove the 5% Terg-a-zyme solution.
6. Repeat steps 4-5 at least three times for a total of at least four washes or as needed.
8. Remove the 100% ethanol. Rinse the hair with 1.0 ml of deionized H2O. Recap tube, agitate several times and remove water.
9. Prepare reagent blank for each sample as follows: Add 187µl of Extraction buffer to micro tissue grinder. Briefly simulate
grinding. Transfer the Extraction buffer to a labeled 1.7 ml microcentifuge tube
10. To the same micro tissue grinder add 187µl of extraction buffer and place hair(s) in the micro tissue grinder.
12. Transfer the solution into a labeled 1.7 ml microcentrifuge tube.
13. Repeat steps 9-12 for all hairs.
14. Add 5µl of Proteinase K and 8µl of DTT to each reagent blank and sample tube.
15. Incubate at 56 °C for a minimum of 2 hours.
16. Add 200 µl phenol/chloroform/isoamyl alcohol (25:24:1). Mix thoroughly. Spin for 2 minutes at 10,000-15,000 rpm in a
microcentrifuge. Transfer upper aqueous layer to a sterile, labeled microcentrifuge tube. If necessary, repeat extraction with
phenol/chloroform/isoamyl alcohol until the interface is clean. Dispose of phenol waste in the appropriate waste container.
17. Add 200 µl n-butanol. Mix thoroughly. Centrifuge 2 minutes at 10,000-15,000 rpm in a microcentrifuge. Remove and discard
most of the n-butanol upper layer into the appropriate waste container.
19. Add 1.5 ml TE buffer to the sample reservoir of the Centricon-30® . Recap before transfer to the chemical fume hood.
20. Transfer the lower aqueous layer to the sample reservoir of the Centricon-30® . Avoid pipetting any residual n-butanol. Spin
21. Add 2 ml TE buffer and spin 15-30 minutes at 4000 x g using Centricon-30® or until the buffer has spun through.
22. Transfer retentate to a sterile, labeled, microcentrifuge tube. Adjust final volume to approximately 50 µl. Record total volume.
Comments: ____________________________________________________________________________________________
___________________________________________________________________________________________________________
DNA Form 39 022601
Page 50 of 70
Organic Extraction of DNA from Mounted Hairs
Item Source Lot #

1 M DTT
Proteinase K
Phenol:Chloroform:Isoamyl Alcohol
n-butanol
TE Buffer
# Specimen Amount Extracted Approximate Volume Recovered(µL) Approximate Final Volume(µL)
Specimen #:
Specimen#:
Specimen #:
Specimen #:
Reagent Blank #:
Reagent Blank #:
Reagent Blank #:
Reagent Blank #:
1. Place several drops of the appropriate mounting media solvent around the perimeter of the coverslip and carefully remove.
2. Using forceps, carefully remove hair(s) and place in a sterile, labeled microcentrifuge tube containing 1.0 ml mounting media
solvent. Recap tube.
3. Incubate at room temperature for approximately five (5) minutes. Gently agitate several times during incubation.
4. Carefully remove the mounting media solvent.
5. Add 1.0 ml sterile, deionized water. Gently agitate hair(s) to rinse off the mounting media solvent. Carefully remove water.
7. Clean micro tissue grinders with 10% bleach, water, and ethanol, in that order. Allow to dry completely before using.
8. Irradiate the micro tissue grinders in the ultraviolet crosslinker according to protocol.
9. Carefully remove hair(s) and place in a different sterile, labeled, 1.7 ml microcentrifuge tube.
10. Add 1.0 ml of 5% Terg-a-zyme solution and place the tube in the sonicator for approximately twenty (20) minutes.
11. Remove the 1.7 ml tube from the sonicator and carefully remove the 5% Terg-a-zyme solution.
12. Repeat steps 10-11 at least three times for a total of at least four washes or as needed.
14. Remove the 100% ethanol. Rinse the hair with 1.0 ml of deionized H2O. Recap tube, agitate several times and remove water.
15. Prepare reagent blank for each sample as follows: Add 187µl of Extraction buffer to micro tissue grinder. Briefly simulate
grinding. Transfer the Extraction buffer to a labeled 1.7 ml microcentifuge tube
16. To the same micro tissue grinder add 187µl of extraction buffer and place hair(s) in the micro tissue grinder.
18. Transfer the solution into a labeled 1.7 ml microcentrifuge tube. Repeat steps 15-18 for all hairs.
19. Add 5µl of Proteinase K and 8µl of DTT to each reagent blank and sample tube. Incubate at 56 °C for a minimum of 2 hours.
20. Add 200 µl phenol/chloroform/isoamyl alcohol (25:24:1). Mix thoroughly. Spin for 2 minutes at 10,000-15,000 rpm in a
microcentrifuge. Transfer upper aqueous layer to a sterile, labeled microcentrifuge tube. If necessary, repeat extraction with
phenol/chloroform/isoamyl alcohol until the interface is clean. Dispose of phenol waste in the appropriate waste container.
21. Add 200 µl n-butanol. Mix thoroughly. Centrifuge 2 minutes at 10,000-15,000 rpm in a microcentrifuge. Remove and discard
most of the n-butanol upper layer into the appropriate waste container.
23. Add 1.5 ml TE buffer to the sample reservoir of the Centricon-30® . Recap before transfer to the chemical fume hood.
24. Transfer the lower aqueous layer to the sample reservoir of the Centricon-30® . Avoid pipetting any residual n-butanol. Spin
25. Add 2 ml TE buffer and spin 15-30 minutes at 4000 x g using Centricon-30® or until the buffer has spun through.
26. Transfer retentate to a sterile, labeled, microcentrifuge tube. Adjust final volume to approximately 50 µl. Record total volume.
Location of DNA Extracts _________________________ DNA Form 37 121099
Page 51 of 70
H. Organic Extraction of DNA From Dried Skeletal Remains – Procedure
Special Supplies, Reagents and Equipment: Aluminum Oxide Grinding Stones, Dremel Tool,
Emery Wheel (0.25" thick), Waring Blender, Hammer, Chisel, Mortar, Nutator, Incubator.
1. Record gross weight of bone specimen.
Perform in Bone Sanding Hood
2. Sand all exposed surfaces of bone with a clean aluminum oxide sanding bit fitted to a
rotary tool. Use an Emery Wheel to cut the bone, if necessary. Clean hood appropriately
and change Dremel bit, gloves, and disposable sleeves between each specimen.
3. Obtain approximately 2.0 g of bone specimen. If necessary, break the specimen into 2 or 3
fragments with a clean chisel and hammer in a mortar. Weigh the bone specimen.
Repackage bone specimens not to be used. Place bone fragment in weigh boat and carry to
laminar flow hood.
4. Clean specimen of powdered debris by placing the sanded skeletal fragments into a 50 ml
conical tube containing approximately 25 ml of bottled sterile deionized water. Shake the
tube back and forth several times. Decant into a waste container. Repeat twice.
5. Cover the specimen in the conical tube with 95% ethanol. Shake the tube back and forth
several times. Decant into waste container. Repeat twice. Pour fragments into a labeled
weigh boat and allow to air dry in the laminar flow hood.
6. Place specimen fragments into a clean Waring Blender cup. Carefully place the lid on.
Perform in the Bone Sanding Hood
7. Take sealed blender cup to base unit. Run blender until bone fragments are finely ground.
If sample is not completely ground after one minute, shut off blender, tap the container,
and repeat.
Note: When starting the unit, the bone may become wedged between a blade and the cup
wall. If so, shut off power, remove cup and dislodge bone by tapping or rotating the blade
spindle from below.
8. Pour powder into a clean weigh boat or funnel for transfer to a labeled sterile, preweighed
or tared 15 ml conical tube.
Perform in the General Laboratory
9. Determine the weight of the pulverized bone specimen in the conical tube.
Note: You may stop at this point for the day. Store pulverized bone sample in the -20°C
freezer.
Page 52 of 70
10. Initiate a reagent blank at this time. The reagent blank should be the last specimen
processed for the remaining steps.
Perform in the Laminar Flow Hood
11. Add 3 ml Extraction Buffer (assure that the Extraction Buffer is homogenous and
prewarmed to 56˚C) and 100 µl 20 mg/ml Proteinase K to the specimens and the reagent
blank. Suspend the bone dust thoroughly in the reagents. Incubate overnight at 56°C on a
tilted Nutator ensuring that the reagents do not reach the cap of the tube.
12. Extract with 3 ml Phenol/Chloroform/Isoamyl Alcohol (25:24:1). Mix thoroughly.

Centrifuge 2 minutes at 4950 x g using the Beckman GPR Centrifuge (6400 RPM) or the
IEC Centra MP4 (5000 RPM). Transfer upper aqueous layer to a clean labeled tube.
Repeat extraction with Phenol/Chloroform/Isoamyl Alcohol until the interface is clean, or
a minimum of two times. Dispose of phenol waste in the appropriate waste container.
13. Extract with 3 ml n-Butanol. Mix thoroughly. Centrifuge 2 minutes at 4950 x g (IEC
Centra MP4 or Beckman centrifuge at 5000 RPM). Remove and discard most of the n-
Butanol upper layer into the appropriate waste container.
14. Assemble a sufficient number of Centricon-100 concentrators and label appropriately.
15. Transfer the lower aqueous layer to the corresponding Centricon-100 concentrators.
Note: Try to avoid pipetting any residual n-Butanol.
16. Centrifuge approximately 30 minutes, or longer if necessary, at 1000 x g (2600 RPM)

using the IEC Centra MP4. Discard filtrate.
17. Add 2 ml of sterile TE Buffer to sample reservoir. Centrifuge approximately 30 minutes,

or longer if necessary, at 1000 x g (2600 RPM) using the IEC Centra MP4. Discard
filtrate. Repeat once.
reservoir.
Page 53 of 70
21. Repeat the extraction procedure if at least 0.5 g bone specimen is remaining.
You will need the following worksheets for this procedure:
“Preparation of Dried Skeletal Remains for Extraction” – DNA Form 2

“Extraction of DNA from Dried Skeletal Remains” – DNA Form 3
Page 54 of 70
Preparation of Dried Skeletal Remains for Extraction
Followed Version ____ of the "AFDIL DNA Extraction Manual”
Case Number:_________________________Date/Time:___________________Scientist:_________________________
Weight of Weight of
Specimen Gross Sanded Fragment Powder
Weight Weight to be to be Hood Grinder Comments
(g) (g) Pulverized Extracted # #
(g) (g)
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Item Source Lot #
Distilled Water
Ethanol
1. Remove outer and inner layer of bone with Dremel tool.

2. Obtain a fragment of bone approximately 2.0 grams.
3. Perform in a Laminar Flow Hood: Place bone specimen in a 50 ml conical tube and fill with distilled
water, allowing space at the top for movement. Place cap on tube and gently agitate the bone in the
water. Repeat. Discard water and wash with ethanol. Repeat. Allow bone to dry at room temperature in
the Laminar Flow Hood.
4. Pulverize to a powder using a clean Waring Blender.

5. Transfer bone powder to a labelled 15 ml conical tube.
Comments: ___________________________________________________________________________________
_________________________________________________________________________________________________
_________________________________________________________________________________________________
_________________________________________________________________________________________________
_________________________________________________________________________________________________
Location of Re-packaged Bones _______________________________________________________________________

Page 55 of 70
Extraction of DNA from Dried Skeletal Remains
Version _____ of the "AFDIL DNA Extraction Manual”
Extraction
Item Source Lot #
1-Butanol AFDIL QC
TE Buffer AFDIL QC
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Reagent Blank #:
1. Add 3 ml extraction buffer to pulverized specimen in 15 ml conical tube.

4. Add 3 ml phenol:chloroform:isoamyl alcohol (25:24:1). Mix thoroughly.

5. Centrifuge 2 minutes at 4950 x g (Beckman 6400 rpm/IEC 5000rpm).
6. Transfer upper aqueous layer to a sterile, labelled 15 ml conical tube.
7. Perform steps 4, 5, and 6 a minimum of two times or until the interface is clean.
8. Add 3 ml 1-butanol. Mix thoroughly.
9. Centrifuge 2 minutes at 4590 x g.
10. Add lower aqueous layer from butanol extract to sample reservoir of Centricon-100®.
11. Spin column at 1000 x g (IEC 2600 RPM) for 30-60 minutes or until sample has spun through. Add 2 ml TE
Buffer and spin 30-60 minutes or until TE Buffer has spun through. Discard filtrate and repeat 2 ml TE Buffer
wash.
12. Transfer retentate to a sterile, labelled tube. Adjust the final volume to approximately 100 µl with TE.
Comments: ____________________________________________________________________________________
___________________________________________________________________________________________
_________________________________________________________________________________________________

Page 56 of 70
I. Organic Extraction of DNA from Teeth – Procedure
Special reagents, supplies, and equipment: Dental hand-piece with straight burs (#4 or #6
round), spoon excavator, clamp, sonicator
Powdered tooth: pour tooth powder into a clean, labeled 15 ml conical tube and proceed to
step 10.
Intact tooth with adherent soil or debris
1. Weigh the tooth specimen.
2. Clean specimen by placing the tooth into a 50ml conical tube containing approximately 25
ml of sterile, distilled water. Cap tube securely and place in ultrasonic bath for 1 minute.
Decant into a waste container. Repeat until gross debris or soil is still no longer observed
on external surface of the tooth
Note: Before washing specimen, set aside any dried soft tissue or loose bone adhering to
the tooth as this material may contain a relatively large amount of DNA. This material can
be extracted separately at a later time. Since it may also contain external contaminants,
this material should not be combined with the pulp and/or dentin sample.
3. Rinse tooth thoroughly with 10% commercial bleach and wipe down with a bleach-
moistened sterile 4 x 4 gauze pad. Follow immediately with a wipe down with 95%
ethanol.
4. Place the tooth into a clean weigh boat and allow to air dry under an UV lamp. Proceed to
Step 5.
Note: Allowing weigh boat to air dry may reduce static compared to drying it with a
kim-wipe.
Fractured or severely carious tooth

Intact tooth with minimal adherent soil or debris
Deciduous ("baby") teeth
1. Weigh the tooth specimen.
2. Rinse tooth thoroughly with sterile, distilled water. Dry with a sterile 4 x 4 gauze pad.
Note: Before washing specimen, set aside any dried tissue or loose bone adhering to the
tooth as this material may contain a relatively large amount of DNA. This material can be
extracted separately at a later time. Since it may contain external contaminants, this
material should not be combined with pulp and/or dentin.
3. Wipe down the external surface of the tooth a 10% commercial bleach on a sterile
Page 57 of 70
4 x 4 gauze pad. Follow immediately with a wipe down with 95% ethanol. Do not
irrigate or rinse tooth with either bleach or ethanol.
4. Place the tooth into a cross-linked weigh boat and allow to air dry under a UV
lamp. Proceed to Step 5.
Note: Allowing weigh boat to air dry may reduce static compared to drying it with a
kim-wipe.
Perform in a Bone Sanding Hood (Fan should be OFF) or Fume Hood
5. Using a sterile #4 round bur, carefully cut horizontally through the midline of the tooth,
separating the crown from the roots.
6. Remove any visible pulp from either half of the tooth with a pair of forceps or a spoon
excavator. Place into an appropriate clean and labeled tube and set aside.
Note: Large pieces of pulp should be combined with the pulp and/or dentin drilled from
the inside of the specimen. It should not be combined with any material removed from the
outside of the specimen.
7. Using a dental drill, drill out the dentin from within the crown and the pulp from within the
roots. Be sure to hold the specimen over a weigh boat so that minimal sample is lost. Drill
until all dentin and pulp is removed (approximately 1-3 millimeters), leaving only the
enamel and the outer portion of the roots.
Note: Use caution not to disrupt any morphological features of the crown.
Note: A clamp may be used at this time to hold the specimen.
Note: If the tooth is known to have been exposed to a harsh environment and
preservation of the roots is not paramount, yet suitable sample quantity is a concern, the
root half of the tooth may be placed inside a sterile glove finger-tip and crushed with a
surgical hammer. The resulting powder may be combined with the drilled dentin and pulp
or extracted as a separate sample.
8. Pour the contents of the weigh boat into a clean, labeled tube (or into the tube containing
previously removed pulp, if applicable) using caution as static may be present.
Note: The procedure may be stopped at this point for the day. Store drilled pulp and/or
dentin sample (in 15 ml conical tube) at -20ºC.
9. Repackage any remaining portions of the tooth specimen and store as evidence.
10. Add 3 ml of extraction buffer (prewarmed to 56ºC) and 100 µl proteinase K (20 mg/ml) to
each specimen and the reagent blank. Mix thoroughly. Initiate reagent blank at this time.
11. Incubate at 56ºC overnight.
Page 58 of 70
12. Add 3 ml phenol/chloroform/isoamyl alcohol (25:24:1) to extract. Mix thoroughly. Spin 2

minutes at 4950 x g using the Beckman GPR Centrifuge (6400 RPM) or the IEC Centra
MP4 (5000 RPM). Transfer upper aqueous layer to an appropriate sterile, labeled tube.
Repeat extraction with phenol/chloroform/isoamyl alcohol until the interface is clean.
Dispose of phenol waste in the appropriate waste container.
13. Add 3 ml of n-butanol. Mix thoroughly. Spin 2 minutes at 4950 x g using the Beckman
GPR Centrifuge (6400 RPM) or the IEC Centra MP4 (5000 RPM). Remove and discard
most of the n-Butanol upper layer into the appropriate waste container.
14. Pre-assemble and label a sufficient number of Centricon-100® concentrators.
15. Transfer the lower aqueous layer to the sample reservoir of the Centricon-100®. Avoid
minutes or until sample has spun through.
16. Add 2 ml of TE buffer and spin 15-30 minutes at 1000 x g or until the buffer has spun
through. Discard filtrate and repeat TE buffer wash.
reservoir.
“Organic Extraction of DNA from Teeth” – DNA Form 182
Page 59 of 70
Extraction of DNA from Teeth
Version _____ of the "AFDIL DNA Extraction Manual" SOP
Extraction
Item Source Lot #
1-Butanol AFDIL QC
TE Buffer AFDIL QC
® Amicon
Centricon-100
Ethyl Alcohol
Sterile, Distilled Water
# Specimen Volume Recovered (µl) Final Volume (µl) Weight of Tooth (g)
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Reagent Blank #:
1. Add appropriate amount of extraction buffer to pulverized specimen in tube.

2. Add appropriate amount of 20 mg/ml proteinase K.
4. Add appropriate amount of phenol:chloroform:isoamyl alcohol (25:24:1). Mix thoroughly.

5. Centrifuge 2 minutes at appropriate speed.
6. Transfer upper aqueous layer to a clean tube.
8. Add appropriate amount of N-butanol. Mix thoroughly.
9. Centrifuge 2 minutes at at appropriate speed.
10. Add lower aqueous layer from butanol extract to sample reservoir of Centricon-100®.
11. Spin column at 1000 x g (IEC 2600 rpm) for 15-30 minutes or until sample has spun through. Add 2ml TE buffer
and spin 15-30 minutes or until the buffer has spun through. Discard filtrate and repeat 2ml TE buffer wash.
12. Transfer retentate to a sterile, labelled microcentrifuge tube. Adjust the final volume to approximately 100µl with
TE.
Comments: ____________________________________________________________________________________
_________________________________________________________________________________________________
_________________________________________________________________________________________________
_________________________________________________________________________________________________
Location of DNA Extracts _________________________________________________________________________

Page 60 of 70
J. Organic Extraction of DNA from Human Nail Material – Procedure
Special Supplies, reagents and equipment: Acetone, Dithiothreitol (DTT) - 1 M, Razor Blade,
Sodium Dodecyl Sulfate (SDS) - 10%, Sonicator, Hot/Stir Plate, Heat Block
1. Obtain nail specimen (approximately 2.5 mg).
2. Transfer nail specimen to a sterile, labeled microcentrifuge tube.
In the General Laboratory
3. Weigh nail specimen and record the mass.
Perform in the Laminar Flow Hood (except vortexing, sonicating, and boiling)
Option: If nail specimen is coated with gold-palladium, then add 1 ml 10% commercial bleach.
Vortex for 2 minutes. Remove bleach.
Option: If nail specimen is coated with nail polish, then add 1 ml acetone. Vortex for 2 minutes.
Remove acetone.
4. Add 1 ml sterile, deionized water. Vortex for 2 minutes. Remove water. Repeat as
necessary to clean surface of nail (at least 2 times).
5. Add 1 ml 10% SDS. Ultrasonicate for 10 minutes. Remove SDS solution.
6. Add 1 ml 10% commercial bleach. Vortex for 2 minutes. Remove bleach.
7. Add 1 ml sterile, deionized water. Rinse nail specimen thoroughly. Remove water.
Repeat three times.
8. Add 1 ml sterile water. Boil for 5 minutes (fresh nail) or 20 minutes (aged nail). Remove
water.
9. Cut nail specimen into small fragments using a razor blade. Transfer nail fragments to a
sterile, labeled microcentrifuge tube.
10. Initiate a reagent blank at this time. This specimen must be carried through the extraction
procedure and assayed in parallel with the nail fragments. The reagent blank should be the
last specimen processed for the remaining steps.
11. Add 500 µl extraction buffer (assure that the extraction buffer is homogenous and
prewarmed to 56˚C), 20 µl 1 M DTT, and 12.5 µl 20 mg/ml proteinase K to the nail
fragments and the reagent blank. Incubate for approximately 3 hours or overnight at 56ºC.
Page 61 of 70
12. If the nail fragments have not completely dissolved, then add 20 µl 1 M DTT and 12.5 µl
20 mg/ml proteinase K to both the sample as well as the reagent blank. Incubate for
approximately 3 hours or overnight at 56°C. Repeat until the nail fragments have
completely dissolved or a maximum of 5 times.
Perform in the Chemical Fume Hood
13. Add 500 µl phenol/chloroform/isoamyl alcohol (25:24:1). Mix thoroughly. Spin 5

minutes at 12,000 rpm using the Sorvall MC 12V. Transfer upper aqueous layer to a
sterile, labeled microcentrifuge tube. Repeat, if necessary, until the interface is clear.
14. Add 500 µl butanol. Mix thoroughly. Spin 5 minutes at 12,000 rpm using the Sorvall MC
12V. Remove and discard butanol layer.
15. Assemble a sufficient number of Centricon-100® columns and label appropriately. Add
2.0 ml TE buffer to each column.
16. Transfer lower aqueous layer to labeled Centricon-100 column.
17. Spin at least 15 minutes at 1000 x g using the IEC Centra MP4 (2600 rpm). Discard
filtrate.
18. Add 2.0 ml TE buffer to the Centricon. Spin at least 15 minutes at 1000 x g using the IEC
Centra MP4 (2600 rpm). Discard filtrate.
microcentrifuge tube. Rinse the Centricon filter by adding an aliquot of TE Buffer to
sample reservoir, pipette up and down several times. Add this liquid to the labeled
microcentrifuge tube and resuspend extract to a final volume of approximately 100 µl.
reservoir.
"Preparation of Nails for Extraction" - DNA Form 43

"Extraction of DNA from Nails" - DNA Form 44
Page 62 of 70
Preparation of Nails for Extraction
Followed Version ______ of “AFDIL DNA Extraction Manual”
Specimen Gross Weight (mg) Comments
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Item Source Lot #
10% Commercial Bleach
Acetone
Distilled Water
10% SDS
1. Obtain a nail specimen of approximately 2.5 mg.

2. Remove gold-palladium or nail polish as necessary:
gold-palladium: Add 1 ml 10% commercial bleach. Vortex for 2 minutes. Remove bleach.
nail polish: Add 1 ml acetone. Vortex for 2 minutes. Remove acetone.
3. Add 1 ml distilled water. Vortex for 2 minutes. Remove water. Repeat as necessary.
4. Add 1 ml 10% SDS. Ultrasonicate for 10 minutes. Remove SDS solution.
5. Add 1 ml 10% commercial bleach. Vortex for 2 minutes. Remove bleach.
6. Rinse thoroughly with distilled water. Remove water.
7. Add 1 ml distilled water. Boil for 5 minutes (fresh nail) or 20 minutes (aged nail). Remove water.
8. Cut specimen into small fragments using a razor blade.
9. Transfer fragments to a sterile, labelled microcentrifuge tube.
Comments: ____________________________________________________________________________________
_________________________________________________________________________________________________
_________________________________________________________________________________________________
_________________________________________________________________________________________________
Location of Re-packaged Nails _________________________

Page 63 of 70
Extraction of DNA from Nails
Followed Version ______ of the “AFDIL DNA Extraction Manual”
Extraction
Item Source Lot #
1-Butanol AFDIL QC
TE Buffer AFDIL QC
DTT AFDIL QC
Sterile Water AFDIL QC
Centricon Purification
# Specimen Volume Recovered(µl) Final Volume(µl) Comments
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Reagent Blank #:
Perform all steps for each specimen (nail fragments) and the reagent blank.
1. Add 500 µl extraction buffer to nail fragments in microcentrifuge tube.

2. Add 20 µl of 1 M DTT.
3. Add 12.5 µl of 20 mg/mL Proteinase K.
4. Incubate at 56oC for approximately 3 hours or overnight. If nail is not dissolved, repeat steps 2-4.
5. Add 20 µl of 1 M DTT.
6. Add 12.5 µl of 20 mg/mL Proteinase K.
7. Incubate at 56oC for approximately 3 hours or overnight. Repeat Steps 5-7 until nail fragments have completely
dissolved or a maximum of 5 times.
8. Add 500 µl phenol:chloroform:isoamyl alcohol (24:24:1).

9. Spin 5 minutes at 12,000 rpm in the Sorvall MC 12V.
10. Transfer upper aqueous layer to a sterile, labelled microcentrifuge tube.
11. Repeat Steps 8-10 until interface is clean.
11. Add 500 µl butanol.
12. Spin 5 minutes at 12,000 rpm in the Sorvall MC 12V.
13. Add 2.0 ml of TE buffer to a Centricon-100.

14. Add lower aqueous layer from butanol extract to sample reservoir of Centricon-100.
15. Spin column at 1000 x g (IEC Centra MP4 2600 rpm) for 15 minutes or until sample has spun through. Discard
filtrate and repeat 2 ml TE Buffer wash.
16. Transfer retentate to a sterile, labelled tube. Adjust the final volume to approximately 100 µl with TE.
Comments: ____________________________________________________________________________________
Page 64 of 70
K. Organic Extraction of DNA from Urine – Procedure
1. Agitate urine sample gently to evenly suspend cells and any precipitate present. Pipet 10
ml into a labeled 15 ml conical tube.
2. Spin in centrifuge 30 minutes at 5000 rpm (maximum speed) to pellet cells. Carefully
decant supernatant without disturbing pellet.
3. Add 3 ml extraction buffer (prewarmed to 56°C) and 25 µl proteinase K (20 mg/ml).

Vortex briefly to mix.
5. Add 3 ml phenol/chloroform/isoamyl alcohol. Shake the tube vigorously by hand or vortex

for 30 seconds to achieve a milky emulsion in the tube. Spin the tube for 3 minutes at
5000 rpm in IEC centrifuge.
6. Transfer upper aqueous layer to a labeled 15 ml tube. Repeat extraction with 3 ml

phenol/chloroform/isoamyl alcohol until the interface is clean. Dispose of phenol waste in
the appropriate waste container.
7. Extract with 3 ml n-Butanol. Mix well and centrifuge 3 minutes at 5,000 rpm in an IEC
centrifuge.
8. Assemble a sufficient number of Centricon-100® concentrators in the laminar flow hood

and label appropriately.
9. Transfer the lower aqueous layer to a Centricon-100. Avoid pipetting any residual n-
Butanol. Centrifuge for approximately 15 minutes, or longer if necessary at 1000 x g (IEC
10. Add 2 ml of sterile TE Buffer to a sample reservoir. Centrifuge for approximately 15

microcentrifuge tube. Adjust to a final volume of approximately 100 µl with TE.
reservoir.
Page 65 of 70
"Organic Extraction of DNA from Urine Specimens”/DNA Form 15
Page 66 of 70
Organic Extraction of DNA from Urine Specimens

Extraction
Item Source Lot #
1-Butanol AFDIL QC
TE Buffer AFDIL QC
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Specimen #:
Reagent Blank #:
1. Add approximately 10 ml urine to a labeled 15 ml conical tube.

2. Spin 30 minutes at 5000 rpm (maximum speed) to pellet cells. Remove supernatant and discard.
3. Add 3 ml extraction buffer and 25 µl 20 mg/ml proteinase K. Vortex briefly.
5. Add 3 ml phenol/chloroform/isoamyl alcohol. Vortex.
6. Spin 3 minutes at 5000 rpm.
7. Transfer upper aqueous layer to a labeled 15 ml conical tube.
8. Repeat steps 5, 6 and 7 until interface is clean.
9. Add 3 ml n-butanol. Mix thoroughly. Spin 3 minutes at 5000 rpm.
10. Assemble and label a sufficient number of Centricon 100 concentrators.
11. Transfer the lower aqueous layer to the sample reservoir.
12. Spin column at 1000 x g (IEC 2600 rpm) for 15-30 minutes or until sample has spun through. Discard filtrate.
13. Add 2 ml TE buffer and spin at 1000 x g (IEC 2600 rpm) for 15-30 minutes or until sample has spun through. Repeat.
14. Transfer retentate to a labeled 1.7 ml tube. Adjust volume to approximately 100 µl.
Comments:_______________________________________________________________________________________________________
_________________________________________________________________________________________________________________

DNA Form 15 012601
Page 67 of 70
VII. REFERENCES
Anderson, T.D., Ross, J.P., Lee, D.A., Roby, R.K., Canik, J.J., Holland, M.M., “A Validation
Study for the Extraction and Analysis of DNA from Human Nail Material and it’s Application to
Forensic Casework”, Journal of Forensic Sciences, JFSCA, accepted January 1999.
Armed Forces DNA Identification Laboratory, "Effect of Ultraviolet Irradiation on DNA

Contamination and PCR Amplification Efficiency," Validation Folder, 1996.
FBI Laboratory, PCR-Based Typing Protocols, August 1, 1994.
Fisher, D.L., Holland, M. M.,Mitchell, L., Sledzik, P.S., Wilcox, A.W., Wadhams, M., and
Weedn, V.W.. (1993) “Extraction , Evaluation, and Amplification of DNA from Decalcified and
Un-decalcified United States Civil War Bone," Journal of Forensic Sciences, JFSCA, 38(1): 60-
68.
Forsthoefel, K.F., Papp, A.C., Snyder, P.J., Prior, T.W., (1992) “Optimization of DNA
Extraction from Formalin-Fixed Tissue and it’s Clinical Application in Duchenne Muscular
Dystrophy, AJCP, 98(1): 98-104.
Hagelberg, E. and Clegg, J., (1991) “Isolation and Characterization of DNA from
Archaeological Bone, Proc. of Roy. Soc. of London, 224, 45.
Heller, M.J., Burgart, L.J., TenEyck, C.J., Anderson, M.E., Grenier, T.C., Robinson, R.A.,
(1991) “An Efficient Method for the Extraction of DNA from Formalin-Fixed, Paraffin-
Embedded Tissue by Sonication”, Biotechniques, 11(3): 372-377.
Hochmeister, M., Budowle, B., Borer, U., Eggman, U., Comey, C., and Dirnhofer, R., (1991).
“Typing of Deoxyribonucleic Acid (DNA) Extracted from Compact Bone from Human
Remains”, Journal of Forensic Sciences, JFSCA, 36:1649-1661.
Holland, M.M., Fisher, D.L., Mitchell, L.G., Rodriquez, W.C., Canik, J.J., Merril, C.R., and
Weedn, V.W.. (1993) "Mitochondrial DNA Sequence Analysis of Human Skeletal Remains:
Identification of Remains from the Vietnam War," Journal of Forensic Sciences, JFSCA, 38, (3):
542-553.
"Isolation of DNA from Liquid Blood Samples", from Procedures for the Detection of
Restriction Fragment Length Polymorphisms in Human DNA. FBI Laboratory, December 7,
1990.
Lee, H., Pagliaro, E., Berka, K., Folk, N., Anderson, D., Ruano, G., Phil, M., Keith, T., Phipps,
P., Herrin, G., Garner, D., and Gaensslen, R., (1991). “Genetic Markers in Human Bone: I.
Deoxyribonucleic Acid (DNA) Analysis”. Journal of Forensic Sciences, JFSCA 36:320-330.
Sambrook, J., Fritsch, E. F., and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd
edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989.
von Beroldingen, C., Roby, R. K., Sensabaugh, G.F., and S. Walsh, "DNA in Hair," in
Proceedings of the International Symposium on the Forensic Aspects of DNA Analysis, 1989, p.
265.
Page 68 of 70
Wilson, M. R., Polanskey, D., Butler, J., DiZinno, J. A., Replogle, J., and B. Budowle, (1995)
"Extraction, PCR Amplification and Sequencing of Mitochondrial DNA from Human Hair
Shafts," Biotechniques, (18)4: 662-669.
“Standard Operating Procedures and Guidelines for Specimen Selection, Tissue Harvesting, and
Documentation for Sampling Mitochondrial DNA Material from Odontologic Remains”,
Central Identification Laboratory, Hawaii, CILHI LAB SOP #3.2.
Page 69 of 70
VIII. ADMINISTRATIVE REVIEW
Date: ________________ By:________________________________________
Date: ________________ By:________________________________________
Date: ________________ By:________________________________________
Date: ________________ By:________________________________________
Page 70 of 70

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DoD DNA Registry

Office of the Armed Forces Medical Examiner

ARMED FORCES DNA IDENTIFICATION LABORATORY

DNA EXTRACTION MANUAL

Adopted By: ______________________________________ Date: _______________

Reviewed By: ______________________________________ Date: _______________

Chelex® 100 is a chelating resin composed of styrene divinylbenzene copolymers containing

II. BIOLOGICAL SPECIMENS

Whole Blood and Bloodstains

III GENERAL REAGENTS, SUPPLIES AND EQUIPMENT

Chelex 100® (Bio-Rad) in sterile, deionized water. Make fresh daily.

IV. QUALITY ASSURANCE

1. Extraction of specimens is to be performed in the following designated laboratories:

whole blood specimens and bloodstains Lab 3

6. Microcentrifuge tubes (closed) should be irradiated in an ultraviolet crosslinker with 2

8. Any supplies or equipment taken from a post-amplification room to a pre-amplification

13. Use aerosol-resistant pipet tips for all liquid transfers.

3. Add 3 - 5 µl of whole blood or a piece of bloodstained material (approximately a 1/8"

5. Spin in microcentrifuge for 3 minutes at 10,000 - 15,000 rpm.

7. Resuspend the pellet in 5% Chelex to a final volume of 200 µl.

8. Incubate at 56o C for at least 30 minutes.

9. Vortex at high speed for 10 seconds.

10. Incubate in a boiling water bath for 8 minutes

11. Vortex at high speed for 10 seconds.

12. Spin in microcentrifuge for 3 minutes at approximately 10,000 - 15,000 rpm.

You will need the following worksheet for this procedure:

Item Source Lot #

Specimen Type Amount

1. Add 1 ml sterile, distilled water to microcentrifuge tube.

2. Dissect a piece of tissue or bone approximately 1 - 2 mm square.

4. Incubate at 56o C for 30 minutes.

5. Vortex at high speed for 5 - 10 seconds.

6. Incubate in a boiling water bath for 8 minutes.

7. Vortex at high speed for 5 - 10 seconds.

8. Spin in microcentrifuge for 3 minutes at 10,000 rpm.

9. The sample is now ready for quantitation (optional) and amplification.

You will need the following worksheet for this procedure:

Item Source Lot #

Specimen Type Amount

1. Add 200 µl of 5% Chelex®-100 to a 1.5 ml tube.

1.7 ml tube = lower portion of Spin-EASE

Dried swabs and dried stains:

1. Dried swabs proceed to step 3.

4. Add 1.0 ml sterile water to the tube.

5. Vortex at high speed for 10 seconds.

6. Incubate at room temperature for 30 minutes.

Swabs preserved in isopropanol:

12. Vortex at high speed for 10 seconds.

17. Remove and discard all but 50 µl of supernatant.

NOTE: When pipetting Chelex solutions the resin beads must be

19. Incubate at 56˚ C for 15 - 30 minutes.

20. Vortex briefly.

21. Incubate in a boiling water bath for 8 minutes.

22. Vortex briefly.

23. Spin in microcentrifuge for 3 minutes at 12,000 - 15,000 rpm.

You will need the following worksheet for this procedure:

Case Number: __________________________ Date:_____________ Scientist:________________

Item Source Lot #

Specimen Amount Extracted Comments

Location of DNA Extracts____________________________________

1.7 ml tube = lower portion of Spin-EASE

4. Vortex briefly and incubate at room temperature for 30 minutes.

7. Spin the sample in a microcentrifuge for 1 minute at maximum speed (10,000-

Adopted By: ________________________ Date: _

Reviewed By: ________________________ Date: _

Case Number: ____________________ Date:_ Scientist:__________

Case Number: ______________ Date:_ _Scientist:__________________________