MTB DNA EXTRACTION KIT

MTB DNA EXTRACTION KIT 48 Reactions 96 Reactions

Reagent Volume Volume

Lysis Buffer 5 ml 10 ml

Neutralization Buffer 5 ml 10 ml

Lysis Buffer BB3 15 ml 30 ml

Wash Buffer BBW (Concentrate) 19 ml 19 ml x 2

Wash Buffer BB5 (Concentrate) 13 ml 13 ml X 2

Elution Buffer BBE 15 ml 30 ml

MTB DNA Spin Columns 50 100

COLLECTION TUBES 50 100

 PREPARATION OF WORKING SOLUTIONS

Wash Buffer BBW: Add the below indicated volume of ethanol (96 – 100 %) to Wash Buffer
BBW Concentrate. Mark the label of the bottle to indicate that ethanol was added. Store
Wash Buffer BBW at room temperature (18 – 25° C) for up to one year.

Format Volume of BBW Volume of Ethanol to be added

48 rxns 19ml 25 ml to each bottle

Wash Buffer BB5: Add the below indicated volume of ethanol (96 – 100 %) to Wash Buffer
BB5 Concentrate. Mark the label of the bottle to indicate that ethanol was added. Store
Wash Buffer BB5 at room temperature (18 – 25° C) for up to one year.

Format Volume of BB5 Volume of Ethanol to be added

48 rxns 13 ml 30 ml to each bottle

• Preheat Elution Buffer BBE to 70⁰C

Note: Before the DNA isolation, viscous samples must first be concentrated and neutralized,
respectively. For the analysis of sputum and PUS, we recommend NALC-NaOH
decontamination; after a final centrifugation, the bacteria pellet can be used for the DNA
isolation.

Procedure:

1. Add 1 volume of NALC solution to 1 volume of the sample. Incubate for 10-15 minutes
at room temperature while agitating gently.
100 ml NALC solution contains:
• 50 ml 2.94% sodium citrate
• 50 ml 4% sodium hydroxide
• 500 mg N-acetyl-L-cysteine
2. When sample attains the desired fluidity add 2 volumes of sterile phosphate buffer (pH
6.8). Centrifuge the specimen tubes at 4,000 rpm for 15 minutes.
3. Discard the supernatant and continue with step 1 of the MTB DNA Extraction Kit
protocol.
S.No. Sputum,Bronchial Washings and PUS
1. Add 1 volume of NALC solution to 1 volume 1 volume Sample+1
of the sample. Incubate for 10-15 minutes at volume NALC-NaOH +
room temperature while agitating gently. Incubate for 10-15
minutes at RT.
2. When sample attains the desired fluidity add 2 Add 2 volumes of PBS.
volumes of sterile phosphate buffer (pH 6.8). +
Centrifuge the specimen tubes at 4,000 rpm for Centrifuge at 4000 rpm
15 minutes. for 15 minutes.
3. Discard the
Discard the supernatant
supernatant.
4. Add 100 µl of Lysis buffer and proceed with the DNA Extraction Protocol.

S.No. CSF, Urine and other body fluids

1. Centrifuge the samples at 12000 rpm for 5 min and Centrifuge at 12000rpm
discard the supernatant for 5 min
2. Add 100µl of Lysis Buffer to the pellet and proceed Follow DNA Extraction
with step 1 in the given protocol. Step 1.

S.No. Blood Samples

1. Centrifuge at 2000rpm
Centrifuge the blood samples (collected in EDTA
for 15 min at 4°C.
vials) at 2000 rpm for 15 min at 4°C for the
+
separation of plasma. Collect the supernatant in a
Take the supernatant in
new micro centrifuge tube.
a fresh vial.
2. Centrifuge at 12000
Centrifuge the plasma samples at 12000 rpm for 10
rpm for 15 min.
min at room temperature and discard the
+
supernatant, leave around 100µl in the tube if any
Discard the
pellet is not visible.
supernatant.
3. Add 100µl of Lysis Buffer to the pellet and proceed Follow DNA Extraction
with step 1 in the given protocol. Step 1.
 INSTRUCTIONS FOR DNA EXTRACTION

Step Description Process

Sample
SAMPLE LYSIS:
+ 100µl Lysis Buffer
1 • Add 100µl Lysis Buffer to the pellet.
Incubate at boiling water
• Incubate at boiling water bath (95°C) for 15 min.
bath (95°C) for 15 min
+
SAMPLE Neutralization:
100µl Neutralization buffer
2 • Add 100µl Neutralization buffer to the samples and mix
Vortex & centrifuge 2 min at
well and centrifuge 2 min at 10,000 rpm.
10,000 rpm
ADJUST DNA BINDING CONDITIONS:
200 μl of supernatant
• Transfer 200 μl of supernatant in to a new 1.5 ml
+
microcentrifuge tube.
250 μl Buffer BB3
3 • Add 250 μl of Buffer BB3 and 250 μl of ethanol (96-
+
100%) to samples and vortex the mixture vigorously
250µl Ethanol
(10-20 s).
Vortex
BIND DNA:
• Pipette the mixture from previous step into the Spin Load lysate into a column
4 Column placed into a 2 ml collection tube. Centrifuge Centrifuge 1 min, 10,000
for 1 min at 10,000 rpm. rpm
• Discard flow-through.
WASH SILICA MEMBRANE:
1st Wash +
• Add 500 μl Buffer BBW. Centrifuge 1 min at 10,000 rpm. 500 μl Buffer BBW
Discard flow-through and place the column back into centrifuge 1 min, 10,000rpm
5
the Collecting tube. +
2nd Wash 500 μl Buffer BB5
• Add 500 μl Buffer BB5. Centrifuge 1 min at 10,000 rpm. centrifuge 1 min, 10,000rpm
Discard collecting tube with flow-through.
DRY SILICA MEMBRANE:
• Place the column into a new collection tube and
5 centrifuge 3 min, 12,000 rpm
centrifuge for 3 min at 12,000rpm. Residual ethanol is
removed during this step.
ELUTE HIGHLY PURE DNA:
• Place the column in a new 1.5 ml microcentrifuge tube
50 μl Buffer BBE (70ºC)
and add 50 μl prewarmed elution Buffer BBE (70°C).
6 Incubate 1 min
Dispense buffer directly onto the silica membrane.
centrifuge 1 min, 12,000rpm
Incubate at room temperaturefor 1 min. Centrifuge 1
min at 12,000 rpm.
Note: Briefly centrifuge the 1.5 ml tube to remove drops from the inside of the lid after each
incubation and vortexing step.