Huey Lee
SID# 13073096

Southern Blot


Purpose:
In this experiment, we used a technique called Southern blot to probe the genomic DNA of a 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. Since small pieces transfer more efficiently than large ones, we will first cut the DNA with restriction enzymes. Also, single stranded DNA stuck to the membrane, so the DNA was denatured. Then after neutralizing the gel, the DNA was moved from the gel to the membrane by a wicking mechanism, and a stack of dry paper towels provided the wicking power. 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 precipitated the DNA by adding 10M ammonium acetate and ethanol. We spooled the DNA 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. The DNA was then run on a gel to check for quality.
After quality checking our genomic DNA, it was 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.
Then, the DNA in the gel was denatured so that it could be transferred efficiently onto the nylon filter. The gel was equilibriated 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 gel 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 BanH 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 .5pg, 5 pg, and 50 pg 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. Bluish, purplish bands appeared on both sides of the filter. 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 not 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. 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. This also 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 band 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 genmonic 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.
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 that suggests that our southern blot was successful. 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 seem 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.
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.



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