Problems with Southerns!

[Gayatri Mani]

Dear Roger,
Thanks for your advise on Southerns.Perhaps I am using too much probe.I used 
3 microlitres of a concentration of 13.1 nanograms/microlitre.So you are 
saying picogram quantities for a genomic southern.Any figures? Could you 
please shed some more light on how to work out the amount needed.It was 
suggested to dilute the probe using an equation from maniatis to figure out 
the sensitivity of the assay,because of the size of the mammalian 
genome...estimated at
2x10^9.The probe fragment is 511bp long.Would I still cut the 
plasmid,isolate the gene fragment,purify and then use it in dilutions or 
not?I add 10 micrograms of genomic DNA to the blot.If this information is 
useful could you please guide me a little more.I would much appreciate 
it.And also,I am using EcoR1 because it produces fragments of about 
2000bp,2500bp,5600bp and 7500bp.Only the 2500 and 5600bp seem to show any 
signs of coming up after 10 days of exposure,however the bands are too 
faint! Would using a lot of probe be of any use? If I can get 14microlitres 
of the DNA in one prep,should I dump the whole lot in to label? In that case 
would that alter the amount of the other solutions like nucleotides etc,...?
The acid treatment you said was with HCl.I had never depurinated the gel,but 
was suggested to do so.I do it for 20-30minutes.Does that not sound right?
Sorry to have asked so many questions,but I am desperate to get this sorted 
out.Please reply ASAP.
Thanking you very much in advance..

Regards,
Gayatri


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