Southern blot not working - help (Aug/12/2005 )

I am currently working with Homalodisca Coagulata and I have not had any of my southern blots to work. I have tried loading upto 20ug but it still does not work. The controls work just fine everytime. I have tried to doing genomic PCR and RT PCR and both of them work just fine.

I have also not been able to find any evidence of a southern blot with this insect. The southern blot with another insect worked just fine. For one of the genes I did gene mapping to replace southern blot which worked just fine. But it is hard to do gene mapping for so many genes. I don't know if the genome is too big or if it something else

Has anyone had any experience with that? Please help if you know why this is the case and if there is a way to get around it rather than doing gene walking for every single gene I have.

Nadia

Hi Nadia,

I assume when you say "not working" you mean that you are not getting a band after hybridisation. Do you have an internal positive control? What I mean is, do you re-probe the same blot with a probe sequence that you already know is in the genome or that worked previously? This might tell you whether it is your digestion and size separation or your hybridisation that is failing. In the absence of more info, my money is on the digestion failing, or the gDNA being aggregated or degraded. Did you check that the digested DNA left the wells during electrophoresis and gave a nice smear centred around the expected modal fragment size for your enzyme?

Cheers,

Stu

Hi Stu,

The problem is that the southern blot for this particular insect has never worked for me or for anyone else in my lab. I also do not find any published results for a soutern blot for this insect. So internal positive control is not an option. I have also heard of other labs not being able to do a southern blot on this particular species.
The dna looks pretty well digested. It also does not run off the gel. I do not think degradation is a problem. I do not know about anything else that maybe hindering the hybridization. I am having success with gene walking but I would like to figure out why the southern blot would not work.

Thanks