I'm a graduate student of Sogang University in Korea.
I have performed an experiment of the Southern Hybridization of axolotl
genomic DNA three times a few days before, but I couldn't get any data.
I purified the Genomic DNA from the liver and the muscles of axolotl,
and then treated it with restriction enzymes:BamHI, EcoRI, HindIII.
Each samples were loaded about 50-60 ug per a lane. At this time,
I used 0.7% agarose gel containing EtBr(5microgram/ml)-We usually add
EtBr when making the gel-. The gel was running at 30-50V for 8-12 hours.
I transfered separated DNA to nylon membrane(Hybond N+) using vacuum
transfer method or capillary transfer method. Since no signal was
in vacuum transfer at first time, I tried capillary transfer from that
time because I think that the reason why I couldn't get any result is
because the axolotl genome size is too large to transfer in vacuum
method. And then, in capillary method, I performed a serial procedure
for the complete transfer:depurination, denaturation, neutralization,
after which the transfer was performed for about 24 hours.
The transfered nylon membrane was crosslinked at 150mJ, and dried
at 65C-oven. I made it prehybridized (6X SSC; 50% Formamide;
0.05X BLOTTO)and hybridized with P32-labeled probe at 41C. However,
I couldn't find any signals on membrane. So, I tried the pre -
and hybridization step at 30C, but no signal was detected.
Why any signals don't appear? I also tried to load a small amount of
the sample(10 ug), but the result was same as above. If there is anybody
who succeeded in the Southern of axolotl genomic DNA or who often dealt
with axolotl genome in southern, could you comment me something to be
careful and to be considered important in performing the Southern of
axolotl genomic DNA? This is very Important for me because I am a
freshman in graduate school so I'm poor at performing an experiment
yet. I'll wait for the answer from anybody. Appreciate that.
Lab of Developmental Biology