Huey Lee
SID# 13073096
Southern Blot Analysis of Xenopus Laevis Genomic DNA
Purpose:
In this experiment, we used a technique called Southern blot to probe the genomic
DNA of Xenopus Laevis nuclei for the presence of actin. Our first goal was to precipitate
out the genomic DNA from a suspension of nuclei given to us and run on a gel. Our next
goal was to transfer this DNA onto a nylon membrane. After the DNA transfer, the
membrane is hybridized with a probe, a PCR product from a previous experiment,
labeled with deoxgenin. Later, a colorimetric method is used to see if the actin was
indeed detected in our nuclei DNA.
Procedure: (see Lab Manual)
Starting with the nuclei suspension, first we added proteinase K. Then we induced
a nuclei lyse by adding NaCl, EDTA, and SDS. We then proceeded to precipitate the
DNA by adding 10M ammonium acetate and ethanol. The DNA was spooled out with a
heat sealed pipette and washed it 70% ethanol. Then the DNA was resuspended in TE
and left on the rocker for a few days. It was then run on a gel to check for quality.
After quality checking our genomic DNA, we wanted to transfer the DNA onto a
nylon filter. Since small pieces transfer more efficiently than large ones, the DNA was
first digested with restriction enzymes (EcoRI, HindII, and BamH). Also, from a previous
experiment, a PCR product of Xenopus Laevis actin was obtained and cut with the same
enzymes. These were then run on a gel with a lambda marker. Also, single stranded DNA
stuck to the membrane, so the DNA was denatured. The gel was equilibrated in 0.2M
HCl. This depurinates the DNA. The DNA is then treated with base to break the
phosphodiester backbones, which fragments the DNA into small single stranded pieces.
The gel was then neutralized in NH4Ac. The gel was then ready for the DNA transfer via
southern blot.
The southern blot set up was simple. The gel was stacked on a sponge. Then the
filter was stacked on top of the gel. Any bubbles were rolled out, because the DNA
cannot travel through bubbles. Then blotting paper followed by a large stack of paper
towels and a glass plate was stacked on top. The paper towels provide the wicking action
needed to draw the NH4Ac up and through the gel, providing a medium for the DNA to
travel in. Then the filter was baked in a vacuum for 2 hours.
To visualize the DNA of interest, we hybridized the filter with a specific DNA
probe prepared from our PCR fragment, which was an amplified from actin cDNA. The
probe was non-radioactively labeled with a nucleotide analog with a deoxygenin
molecule attached. To make the probe, random primer synthesis was used. This method
relies on the ability of the Klenow fragment of DNA polymerase I to carry out DNA
synthesis in the presence of a primer (a random hexamer mix) and a template. One of the
deoxynucleotides used in this reaction is labeled with digoxigenin while the other three
deoxynucleotides are unlabeled. The end result of this reaction is a DNA probe that is
labeled to a high specific activity with digoxigenin. The filter was left to hybridize over
night.
A histochemical reaction was then used to produce a color precipitate. The filter
was first put through some chemical washes. Then it was put in a plastic zip lock bag
with color development solution and left until the color developed.
Data & Observations:
After first adding SDS and proteinase K, the solution because very viscous and
stringy. This indicated that the nuclei were lysed and the DNA was becoming less
compact. To check the quality of this DNA, we ran it through a gel. The results can be
found in Fig 1. Lane 7 contains the genomic DNA. It shows one band that has not
traveled very far from the wells.
Then the genomic DNA was cut with restriction enzymes (EcoRI, HindIII, and
BamH) and run on a gel along with our PCR product cut with the same enzymes. This is
shown in Fig 2. Lanes 1 and 13 are lambda markers run with the concentration of 0.5
micrograms/10 microliters. Lanes 2, 3 and 4 contain the genomic DNA cut with HindII,
EcoRI, and BamH respectively. The bands are streaks that run from the well. Lanes 5 and
6 are skipped. Lanes 7, 8, and 9 are our PCR product at 0.5pg, 5pg, and 50pg
respectively. There are no visible bands for these lanes. Lanes 11, 12, and 13 contain the
PCR product cut with the three different restriction enzymes.
The DNA from the gel in Fig 2 was then transferred to a nylon filter via a
southern blot. A slant was cut into the corner of the filter to denote which side contained
the PCR product and a square was cut on the other side to denote which side contained
genomic DNA. After developing the filter in development solution over night, colored
bands appeared on the filter. On each “lane” of the filter, bluish, purplish bands. The
bands on the PCR side were bright and clear, while the bands on the genomic side were
faint. Due to the filter being wrapped in saran wrap, it was hard to photocopy and so no
reproduction could be attached.
Conclusion:
After the lysis of the nuclei, we noticed that the solution was viscous and stringy.
This indicated that our lysis worked and that the DNA was now unpacked. The reason for
this was that SDS is a strong detergent, so it denatured the nuclear membranes causing
the DNA within to be released. Proteinase K digests proteins nonspecifically and so went
the contents of the nuclei were released, it began to digest the histones and other proteins
contain within. At this point, he solution became very viscous because the normally small
compact DNA started to become long and stringy.
We then collected and ran this genomic DNA on a gel along with a lambda
marker. The genomic DNA resulted in a single band that did not travel far from the well.
The distance this band traveled was approximately 1.1 cm. From the Graph 1, the
estimated size of the genomic DNA is 21.2 Kbp. This short distance indicates that the
DNA must be very large because it could not travel through the holes in the agarose gel
quickly. This is good because the genomic DNA should be very large and not be able to
travel very far in a gel. The single band means that there was no shearing when isolating
the DNA from the nuclei. Also, there was no other visible band, suggesting that other
nucleic molecules such as RNA fragments and proteins were not present. The intensity of
the band matched the lambda marker with a concentration of 0.1 microgram/10
microliters meaning the concentration of the genomic DNA is about the same. From this,
we could safely say that the isolated DNA was of good quality and proceeded to the next
step.
After digesting the DNA with restriction enzymes, we ran the DNA along with
PCR product on another gel. The resulting get (fig 2) showed bands for our genomic cuts
but not for our PCR products. The streaks seen in our genomic lanes may be because of
the many different restriction sites that must exist on such a large piece of DNA. There
may be a whole range of fragments created from cutting with enzymes and so the bands
may be so close together, they form streaks. It is unclear why we could not see bands for
our PCR. One reason maybe because the concentration of the PCR was too low and so no
visible bands were seen. If we were to see the PCR bands, they would be farther up the
gel than the genomic DNA because compared to the genomic DNA, the PCR product is
much smaller in size.
The DNA from this gel was then transferred onto a filter and then hybridized with
a probe made from our PCR product. We saw bands on the PCR side of our filter. Since
the probe was made from out PCR product, we expected colored bands to appear on the
PCR side of the filter. This suggests that the southern blot transfer of DNA was
successful and that the genomic DNA was also transferred to the filter. Faint bands were
seen on the genomic side. This is also what we expected. The genomic DNA contained
actin, but it was a very small compared the large size of the genomic DNA. So we were
successful in probing our filter for actin.
Through the southern blot method, we were trying to probe Xenopus Laevis
nuclei for the presence of actin. By isolating the genomic DNA from the nuclei and
running it on a gel, we found that it clean and intact with no random proteins of RNA
fragments still in the mix. We then cut the DNA with restriction enzymes and ran it on
another gel with cut PCR. The DNA from this gel was then transferred via southern blot
on a filter that was hybridized by a deoxygenin labeled probe made from out PCR
product. After running it through a color development solution, the filter show colored
bands, indicating that there was indeed some actin DNA in our genomic DNA and in our
PCR product.