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Anthony Araracap

Period 4 AP Biology
(Lab 7A and 7B Lab Report)
Restriction Digestion and Analysis of DNA (7A) and DNA Fingerprinting (7B)

Background:
Gel electrophoresis is a technique used to analyze fragments of DNA. The restriction enzymes
cut the DNA into numerous fragments. Once fragments are generated, they separate from each other
using gel electrophoresis. E.Coli is bacteria used for light. Bioluminescence is when an organism creates
own light through a chemical reaction, while fluoresce is the emission of light by a substance that has
absorbed light or other electromagnetic radiation of a different wavelength. Ampicillin is an antibiotic used
to kill bacteria.

Purpose:
The purpose of the lab was to cut DNA using restriction enzymes, separate DNA fragments using
electrophoretic means. Also, to determine the number of base pairs in fragments of DNA.
Hypothesis:
(Part A) If prepared digested DNA samples undergo gel electrophoresis, then the number of base
pairs in fragments of DNA can be determined. (Part B) If we insert a plasmid, then the transformation of
colonies will be successful.

Materials:
● Restriction Digestion and Analysis of Lambda DNA kit
● ice bath
● 37 degrees C water bath
● 65 degrees C water bath
● micropipettes
● electrophoresis chamber
● Agar plate containing LB/ No Amp
● Agar plate containing LB/AMP plates (red mark on side)
● Sterile inoculating loop
● Plastic transfer pipettes
● Clear 2.0 mL micro tubes
● Cotton swabs
● Sharpie
● Piece of tape for sealing plates
● waste container
● styrofoam cup with ice
● blue 1.5 mL tube of CaCl2
● clear 2.0 mL tube of plasmid DNA labeled either PM1 or PM2
● hot water bath at 42 degrees C
● starter bacterial plate

Procedure:
● (Followed Quick Guide-Restriction Digestion and Analysis procedures & Bacterial Transformation
procedures)
Data & Observations:
During this lab, several observations were made. First off, each colored microtube was a different
color. Yellow tubes were labeled L for lambda DNA, Violet tubes were labeled P for Pstl digest, Green
tubes were labeled E for EcoRI digest, and Orange tubes were labeled H for HindIII digest. TAE was also
used in the lab as a buffer. Another observation was that the bacteria turned blue due to the fluorescent
protein. Also, the plates had each transformed the bacteria, as multiple colonies were grown, although
not all may have been grown. Also, not all the gels produced were readable. Besides this, the starter plate
had a weird odor for the second part of the lab. Furthermore, when the plates that had produced bacteria
were placed under UV light; each group yielded various colors, from green to pink.

Marker Lambda Lambda Lambda Lambda


DNA (no with PstI with with
enzyme) EcoRI HindIII

Distance Base Distance Base Distance Base Distance Base Distance Base
(mm) Pairs (mm) Pairs (mm) Pairs (mm) Pairs (mm) Pairs

Band 1 15 23130 12 37000 20 7000 18 9000 16 9000

Band 2 16 9416

Band 3 19 6557

Band 4 24 4361

Band 5 36 2322

Band 6 39 2027

Analysis:

Error Analysis:
Throughout this lab several errors may have occurred. For instance, using the incorrect amount
of agarose (0.5g). Another error may have been not mixing the components in the tube in order to get the
liquid at the bottom of the tube, yielding less accurate data. Also, when inserting the DNA into the gel
slots, some may not have been carefully placed in, causing inaccuracy in the ladder.The person
transferring had to have a steady hand and good eyes so that the gel wasn’t poked and the DNA made it
into the chamber without problems. Another error may have been that the wrong DNA samples were
added to the wells, but the right ones were identified and later labeled correctly, out of order. The
amounts of the different solutions could have been distorted. Also, there were many lumps in the agar
poured into the plates. The spreading rod that was used to spread the bacteria onto the agar could have
been too hot when it was used to spread, and killed the bacteria, or while the lid of the petri dish was off,
some other contamination from the room could have infected the bacteria causing different results.
Finally, another place of error could have been in
setting of the bacteria on the plates.

Discussion:
The hypothesis for part A of this lab was “If prepared digested DNA samples undergo gel
electrophoresis, then the number of base pairs in fragments of DNA can be determined” was accepted.
Using the base pairs and distance for the DNA L, P, E, and H were able to be graphed once the lab was
concluded. Once the samples were prepared, they underwent agarose gel electrophoresis in the
refrigerator. After, the movement of the restriction enzymes were observed. The genetic marker in the lab
yielded both large and small fragments (base pairs of 23,130 and 15 mm traveled to 2027 base pairs and
39 mm traveled). Furthermore, the L observed in the gel only had one fragment which was 35,000 base
pairs, the P had another fragment; 7000 base pairs, the E and H also had 1 fragment that was 9000
basepairs. As the distance grew bigger, the base pairs were less.
Then, after each procedure for part B was completed, bacteria was placed in UV light to be
observed. Some of the petri dishes had shown that transformation of bacteria had occurred, thus allowing
the plasmid to survive because it was recombinant.The petri dish with LB/Amp (+) was expected to have
many transformed colonies and the results reigned true. Also, in the colonies, tiny dots of bacterial
colonies that were green could be observed. In the second petri dish with LB/Amp (-) green fluorescent
dots were seen, but significantly less than the first dish. In the last petri dish with LB/No amp (-) only a few
tiny fluorescent green circles present.The hypothesis “If we insert a plasmid, then the transformation of
colonies will be successful” for part B was then accepted because colonies were successfully
transformed, as seen in the actual plate results, thus both hypotheses were accepted for part A and B.

Discussion Questions:
(Pre-Lab [A])
1. a. Restriction enzymes cut DNA into numerous fragments, acting like scissors.
b. DNA has a negative charge and will move in the positive direction in an electrical field.
c. The movement of DNA fragments in the gel is effected by due to the negative charge of DNA.
d. Larger fragments move much slower through the gel and thus on a DNA ladder the larger
portions will be near the top.
e. The genetic marker in this case acts are the control.
1. The largest fragment will be D.
2. The smallest fragment would be B.
3.

-
4. The large fragments would be near the top of the gel since it is more difficult for larger pieces to be
strained through the gel.
5. There would still only be 4 bands present.
6. Fragment D would be heaviest because it is the largest piece of DNA and would have the largest
mass.
7. Each enzyme produces different sizes of restriction fragments that have different patterns, which
suggest different enzymes are cutting at random locations.

(Post-Lab [B])

Plate # #1 LB/Amp (+) (red line) #2 LB/Amp(-)(red line) #3 LM/No Amp(-)(no


red line)

Plasmid Present? YES NO NO

Amp present in plate? YES YES NO

Actual Plate
results(drawing)

Actual Plate specks of bacteria, larger colonies with not much visible,
results(description) some colonies weren’t small colonies possibly tiny specks of
transformed bacteria

1. In plate 1 there were about 800 colonies, plate 2 had about 30, and plate 3 had around 20.
2. The plasmid mix we had was EGFP. The three colors of fluorescent bacteria that were observed
were blue, dark green, and light green.
1. Colonies were able to be different colors due to the green fluorescent protein, which is used for
its bioluminescence; produced when the gene is expressed and energy is transferred to it within
its host.

2. The plasmid was able to enter the cell through the bacteria and plasmid DNA solution with
calcium chloride, which neutralized the negative charges.
3. If a group did not get any transformed colonies it may have been due to the mixture not being
placed in the ice bath quickly enough, or incorrect mixtures of the DNA once the restriction
enzymes were added.