DNA Extraction & Analysis
Kylie Walford
Partners: Alec Lamb & Cameron Schwartz
Section: 003
Purpose: The purpose of this lab was to perform agarose gel electrophoresis of our leaf genomic DNA
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:
Figure 1: Shows the gel electrophoresis and 2kb ladder standard to the far left.
Figure 2: Shows the amount of fragments in the ladder and the size (base pairs) in each fragments as well as the mass.
6
5
Rf Value
4
3
2
1
0
0
1
2
3
4
5
LOG10(MW)
Figure 3: Shows the Rf value verses the log of the molecular weight which can be used to estimate other sample fragment sizes.
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.
2. Genomic DNA is isolated by being cut into smaller fragments of manageable sizes with one or
more specific restriction endonuclease. The RNA initially present has essentially been
“digested” by these restriction enzymes which can take over foreign DNA that may enter the
cell. Ethanol precipitates DNA because DNA is polar and is negatively charged due to its
phosphate group whereas ethanol is non-polar. Therefore when ethanol is placed in the presence
of DNA it becomes insoluble and precipitates out of the solution. I would estimate there is
approximately one hundred micrograms of DNA in one gram of plant tissue.
4. See Figure 3.
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 due to the fact that 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.
c. The estimates of the fragment sizes compare rather well to the known sizes of the
fragments.
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 tea 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.