Genomic S. blot problems
leach at bu.edu
Sun Feb 25 10:02:02 EST 1996
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
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
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...
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
: 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!!!
..... Martin Leach Email:leach at bu.edu
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