Genomic S. blot problems

Karen Ferri kferri at email.unc.edu
Sat Feb 24 23:35:47 EST 1996


Hi everyone!

My third attempt at producing a film of some genomic (not PCR) DNA has 
failed and I am very discouraged.  

I have no probs. with PCR S. blots and what follows is my wash protocol 
for those type of blots:

		(Note:  All washes have 0.1% SDS)
		2X SSC @ RT for 5'
		Repeat above, then
		0.2X SSC @ RT for 5'
		Repeat above, then
		0.2X SSC @ 42 degrees for 15'
		Repeat above

I then check the blots with the counter and if still too hot, I wash at 
65 degrees with 0.1X SSC.

My first attempt with these GENOMIC blots was with someone else's protocol:
I washed with 2X SSC @ 42 degrees for 20' and repeated once.  The result:
bands with a lot of background.  BTW, fresh probe prepared.

Second attempt:  used previously prepared probe and used my PCR blot wash 
protocol and did NOT do the 65 degree wash.  Result: Blank film.  Thought 
maybe it was the probe (we fractionate) that was now bad.

Third attempt: Prepared fresh probe (same DNA source and labeled 
beautifully) and again used my PCR blot wash protocol.  The  result: 
Blank film.  

I have zeroed in on the washes.  PCR DNA is different than genomic DNA.  
Probes would not bind as tightly to genomic as PCR DNA simply because all 
that other DNA is also on the filter.  Therefore, I think the PCR wash 
protocol is simply too harsh for genomic blots.  Can anyone comment on my 
rationale?

Assuming it is the washes, how about washing like I did the first time, 
but adding a third wash of 0.2X SSC @ 42 degrees for 15'?  Wouldn't that 
decrease the high background I got on my first attempt while keeping my 
probe on the filter?

Any and all comments, help, suggestions would be greatly appreciated!!!

TIA
KF



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