Pls help southern blot - (Sep/28/2005 )
I have been suffered from southern blot hybridization for many times. I always got no bands but my marker bands still worked well. I try to increase my gDNA concentation from 5 to 15 ug and digested O/N conventional transfer and hybridization. The result still the same. Pls anybody help?.
Is your probe ok? Can you run a plasmid or some other control DNA (eg plasmid) to test this?
Thank you so much for your suggestion, I used the same DNA fragment, that used for probe synthesis, as a positive control. It gave a good signal. But no gDNA band were seen.
Do you think is it because of my gDNA quality or concentration?.
Check your gDNA......if the DNA is good in quality, then increase the amount of DNA per lane(what model r u working on???) if its mammalian, or any other higher vertebrate species, u must take at least 10-15ug DNA per reaction.
Check an aliqout of the digestion first.
And what is the mode of hybridization, i mean, is it radioactive or non-radioactive??? if using non radioactive methos for a higher vertebrate species, it may be tough to detect a single copy gene.
try the above, or clear out the points so that it becomes easy to suggest further.
Do you know if the sequence you are probing for is an exact match for the sequence of the probe or could their be polymorphisms?
I am also facing same problem as you.
Also have been doing this for quite a long time.
Since I am using P32 method this can really be bad at times
So how long is the probe lenth u are working with ? Also my positive controls like urs are working well . just that i am not able to see any bands.
I am suspecting my restriction digestion.. which might be at fault. by the way what is the size, and genome type that u are determining with the probe ?
best of luck ..
Dear tap14, arin and meeta
Thaks so much for all of your suggestion. My gDNA is fish DNA, I used about 400 bp probe and radioisotope 32P for southern blot hybridization every step fallow conventional method of Sambrook and Russell.
Waiting for all of your suggestions
I also have the same problem: with fish gDNA, p32 method with no results (only smear). i can suggest you to do what i'm doing now: i'm trying to detect the sensitivity of this method to understand wheather it can detect my single copy gene.
Try to prepare serial dilutions of a plasmid containing the probe sequence from 1 ng to 1 fg. Blot them with the probe (that prepared from the same plasmid), and see if your method can detect low concentration of DNA. Than try to calculate how much gDNA you sould load to get the same signal in the same conditions.
tell me if it work out, good luck!!!!
Thanks so much I will try your suggestion