Genomic S. blot problems

gordon allison goa at aber.ac.uk
Mon Feb 26 09:19:05 EST 1996


How good is your genomic DNA?  Have you had a good look at the digests on 
gel before transfer, is it clean of protein and has it cut properly?  If so 
the it should give you good signal if your probe is fresh and homologous.  
The wash protocol which you use should be fine.  Other than that, I don't 
know.
       Gordon.




In article <4goov3$15q3 at bigblue.oit.unc.edu>, kferri at email.unc.edu says...
>
>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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