Lab 4 - DNA Restriction Analysis Lab Report

Turn in ONE lab report per group - thank you!

Each individual should complete the lab 4 knowledge check.

Lab Partner(s): Fatima L., Maria Y.

Reference: Micklos DA, Freyer GA, Crotty DA, DNA Science: A First Course, 2nd Edition,
2003

Objectives:
● Cast, load, and run our own agarose gel
● Perform restriction reactions to analyze DNA

Expected Outcomes/Results (what should have happened if the lab ran exactly as
expected);
● If the λ DNA sample contained a higher proportion of circular molecules, then the
restriction digests with EcoRI and BamHI would yield fewer distinct bands on the gel
○ because the circular form of DNA would combine multiple fragments into larger
bands.
● Technically, our λ DNA restricted with HindIII should look something like the ideal gel
with possible bands at 27491 bp, 23130 bp, 9416 bp, 6557 bp, 4361 bp, 2322 bp, 2027
bp, 564 bp, and 125 bp.

Analysis and Results:

Record where you have loaded each sample using the paper chart next to your gel, as well as the
chart below.

Ideal Gel:
 Linear DNA fragments migrate at rates inversely proportional to the log10 of their molecular
weights. For simplicity’s sake, base-pair length is substituted for molecular weight.

Key: B=BamH1, E=EcoR1, H=HindIII, and “-” indicates that no enzyme was added (undigested
λ phage DNA).

We will use the H lane, which contains HindIII digested λ phage DNA, as the molecular weight
marker (or ladder) in your experiment. Please refer to the chart below for HindIII digest DNA
fragment sizes. Label your gel accordingly, and use these HindIII band sizes to compare to your
restriction digest band sizes.

HindIII digest DNA fragment sizes:

Actual bp

27,491a

23,130a

9,416

6,557

4,361
a
2,322 Pair appears as a single band on the gel.
b
2,027 Band may not be visible in a methylene-blue stained gel
 564b c
Band may not be visible with ethidium bromide staining

125bc c
Band runs off the end of the gel when bromophenol blue is approximately
2 cm from the end of the gel.

Your gel:
We are using Gel __5, 6, 7, 8__ -
(Lane 5: H) (Lane 6: B) (Lane 7: E) (Lane 8: -)

Insert the image of your gel here. Be sure to label lanes and the estimated size of each band. Use
HindIII as your DNA ladder.
Key: B=BamH1, E=EcoR1, H=HindIII, and “-” indicates that no enzyme was added (undigested
λ phage DNA).
(dont)Include a paragraph, make a bulleted list of sizes (greatest to smallest) interpreting the
bands you see in each lane here. For example, what are the sizes of each band in your digest
lane? Refer to the labels you provided for bands in the molecular weight marker lane (HindIII
digest/ladder), indicating the number of base pairs.

Interpretation for EcoR1 restriction digest: (Maria Yayloyan)

Lane E
○ Band 1: 24756 bp
○ Band 2: 21226 bp
○ Band 3: 7421 bp

Interpretation for BamH1 restriction digest: Fatima Laoufir

● Lane B
○ Band 1: 16841 bp
○ Band 2: 12275 bp
○ Band 3: 7233/6770 bp
 ○ Band 4: 6527 bp
○ Band 5: 5626 bp
○ Band 6: 5026 bp

See below for help in this interpretation. An example of how a gel is labeled for publication can
be found at the link in Schoology.

The following diagram shows the linear restriction map of the λ phage DNA genome, however, λ
phage DNA can also exist as a circular molecule. These maps will be helpful as you interpret
your bands for your digest reaction.
As mentioned above, λ phage DNA can exist as both a linear molecule and as a circular
molecule. It can circularize because of single-stranded complementary sites that hydrogen bond
together in the circular form. Commercially available λ phage DNA is likely to be a mixture of
linear and circular molecules (and we are using commercially available λ phage DNA) and more
bands appear on the gel than would be predicted from linear DNA molecules alone. The
existence of both forms also causes other fragments to appear less frequently. For example, the
left-most HindIII site is 23,130 bp from the left end of the linear λ genome, and rightmost site is
4,361 bp from the right end. Since band intensity is directly related to DNA concentration, the
4,361 bp band is faint in comparison to other bands on the gel of similar size. This faint
appearance indicates that some of the DNA molecules are circular-combining the 4,361 bp
terminal fragment with the 23,130 bp terminal fragment to produce a 27,491 bp fragment.
However, the combined 27,491 bp fragment usually runs as a doublet along with the 23,130 bp
fragment from the linear molecule.

Conclusion:
Use the CER framework and your work interpreting the digests for EcoR1 and BamHI to draft a
conclusion for this lab. Pick a simple, straightforward claim about the completeness of your
digestion or whether or not the DNA was circularized. You can back these claims up with
evidence. You should include numbers (ie. band sizes in base pairs) for your restriction digestion
reaction. You can compare your results with the ideal gel. If your results failed, let us know
which lab station’s results you are using as your sample.

You will each individually write a claim and evidence; together, you’ll write the reasoning
explaining and justifying the claim(s) you made.

Each lab partner should submit a lab conclusion (some individual, some partner) in the
space below. Only one partner submits a copy of this report.

Partner 1: Maria Yayloyan

The λ DNA sample restricted with the EcoR1 appears to contain a higher proportion of circular
molecules than linear ones, as the presence of distinct high molecular weighing bands is very
limited.

Based on the expected EcoRI digestion pattern, the linear form of λ DNA should’ve produced
distinct bands at approximately 21,226 bp, 7,421 bp, 5804 bp, 5643 bp, 4878 bp, and 3530 bp..
However, Lane E, the lane with the λ DNA restricted with EcoR1, displayed bands at about
24756, 21226, and 7421 bp. The presence of fewer than expected bands, particularly in the
higher molecular weight range, indicates that circular DNA molecules may have combined
terminal fragments, reducing the number of visible bands in the gel.
Partner 2: Fatima Laoufir
Create a simple, straightforward claim about BamHI and give supporting evidence.
The λ DNA sample restricted with the BamHI indicates the gel electrolysis run was too short.

As the gel electrolysis runs, smaller strands go through the gel towards the positive end faster
than larger ones, as their lowered mass allows them to move more freely. However, if a gen run
is too long, similar DNA fragments can separate more than needed. In this case, the amount of
BamH1 strands contained in the lab’s gel is 6, while the ideal amount of strands is 5. This excess
in bands indicated that the gel electrophoresis continued beyond the needed time.
Together: Summarize your reasoning.

In this lab, we used gel electrophoresis to analyze λ DNA samples that we digested with the
restriction enzymes EcoRI and BamHI. Gel electrophoresis is a technique that separates DNA
fragments based on size through the application of an electric current to a gel matrix (agarose in
this case). The DNA strands, having a negative charge, move towards the positive end of the
current, with shorter strands of DNA moving further, faster. By comparing the various bands
formed by our samples to the molecular weight ladder from the HinDIII digest provided, we
estimated the sizes of the DNA fragments of the λ DNA digested with the two enzymes (EcoRI
and BamHI).

For the EcoRI digest (Lane E), we expected distinct bands at approximately 24756 bp, 21226
bp, and 7,421 bp, assuming that the DNA was in a linear form. However, our gel showed fewer
distinct bands in the high molecular weight range, suggesting that a large portion of the DNA
sample was circular rather than linear. This is because circular DNA can combine terminal
fragments, which results in less and larger fragments, as seen in the sample digested with EcoRI.
Oppositely, the BamHI digest showed fragments that contained more characteristics of linear
DNA. In linear DNA, each restriction site produces a separate fragment, resulting in a
predictable number of bands on the gel that is more than there would be if the DNA was circular.
The presence of the strong band at around 16,841 bp, with additional, more faint, bands, shows
that the DNA in this digest primarily existed in the linear form, as linear molecules do not
combine terminal fragments in the same way circular molecules do. The presence of these more
distinct bands, rather than a single, larger combined fragments, shows that BamHI-digested λ
DNA is more linear..