DNA Extraction & Analysis
Kathleen Nolan and Dylan Catterton
Section: 4
November 6, 2014
Purpose: The purpose of this lab was to perform agarose gel electrophoresis of our leaf genomic
DNA extract, wheat germ extract, along with two different samples of Lambda DNA (one that
has been cut or digested with the restricted enzyme Hind III from Haemophilus influenza, and
one that has not been cut.)
Data:
2kb Cut DNA(w/ Hind III) Uncut DNA S9
S3
Figure 1: Shows the gel electrophoresis and 2kb ladder standard to the far left.
Table 1: Shows the amount of fragments in the ladder and the size (base pairs) in each
fragments as well as the mass.
Log10(No. of Base Pairs)
Rf vs Log10(No. of Base Pairs)
4.5
4
3.5
3
2.5
2
1.5
y = -1.8963x + 3.8447
1
0.5
0
0
0.2
0.4
0.6
0.8
Rf
Figure 2: Shows the Rf value verses the log of the number of base pairs
1
Questions:
1. There are many similarities and differences between agarose electrophoresis of nucleic acids
and polyacrylamide electrophoresis of proteins. However, because agarose forms a weaker,
softer, gel than polyacrylamide, electrophoresis in agarose gels in generally performed in a
horizontal orientation rather than vertical. Another difference is that there is no stacking gel in
agarose electrophoresis. This is due to the greater frictional component of nucleic acids in the gel
versus in solution, such that they focus very quickly at the buffer-gel interface before entering
the matrix. On the other hand, agarose electrophoresis is similar to PAGE in that a loading
dye/treatment solution is added to each sample to increase its density, solution properties, and to
allow visualizing the progress of the electrophoresis. Similarly one always includes molecular
weight standards in one lane for calibration purposes.
3.
ABS260:0.755
ABS280:0.491
ABS260/ABS280=1.5
According to the chart that meant our DNA was 20% DNA and 80% Protein
5. See Figure 2.
Lambda Cut with Hind III
Fragment sizes
Fragment 1: Not on graph
Fragment 2: 10,000 bp
Fragment 3: 6,000 bp
Fragment 4: 3,000 bp
Fragment 5: 2,500 bp
Fragment 6: 1,500 bp
Fragment 7: 1,200 bp
Fragment 8: 300 bp
Known sizes
23,130 bp
9,416 bp
6,557 bp
4,361 bp
2,322 bp
2,027 bp
565 bp
125 bp
a. Hind III restriction enzyme is a type II restriction endonuclease. Unlike type I
restriction enzymes, type II restriction endonucleases perform very specific cleaving of
DNA. Type I restriction enzymes recognize specific sequences, but cleave DNA
randomly at sites other than their recognition site whereas type II restriction enzymes
cleave only at their specific recognition site.
b. Not all of the fragments were observed, this could be because the gel may not have
run for long enough and the sample fragment size was too small or because the fragments
were too large to pass through the gel. There was one fragment not seen because it was
larger than our 2kb ladder measured. Three of the fragments were nearly invisible but
were faintly there.
c. The estimates of the fragment sizes compare pretty well to the known sizes of the
fragments except for a few outliers.
Conclusion:
In conclusion in this lab we were able to perform an electrophoresis and compare the known
sample ladder to an unknown sample allowing us to estimate fragment sizes using a graph made
from collected data. We made one of the samples run through the gel via DNA extraction of
young pea leaf tissue. We used restriction endonucleases and cold ethyl alcohol for the DNA
extraction. Two samples of lambda DNA were also run through the gel and the 2kb ladder was
used to compare to Hind III to estimate its fragment sizes.