Genomic S. blot problems

[martin LEACH]

Hi Karen,

several questions.....

what are you blocking your blots with?
Do you use Denhardts? or Blotto? (blotto=non-fat milk...see maniatis)

furthermore, do you use sonicated herring/salmon sperm dna...is it fully
sonicated? do you denature it 10' at 95C before adding it to your hyb
/prehyb solution...

do you have a positive control? put a 5-100ng of the plasmid containing your
probe on a separate blot / in a lane a couple of lanes away from your sample..

what is your probe? 
When I was using some probe a couple of years ago...it had some human
repititive 
DNA sequence in it and I had to block my blots with sonicated human DNA
in the prehyb and hyb steps....to reduce background binding...

just wondering....

Martin

Karen Ferri (kferri at email.unc.edu) wrote:
: 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

--
.....          Martin Leach                Email:leach at bu.edu
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