hybridization problems

[chai_z at wehi.edu.au]

In article <5OCT92.10175724 at wums.wustl.edu>, wetsel_r at wums.wustl.edu writes:
> In a previous article, Klaus.Matthaei at anu.edu.au wrote:
>>>To molecular biologists:
>>>I am having a problem in probing Northern blot membranes ans slot blot 
>>>membranes (Hybond N+). Sllight hazy background is evident on the 
>>>membranes, suggesting that the probe is OK.  However, where the RNA is 
>>>applied in slot blots or where it has migrated to in Northerns appears 
>>>white.  The image looks like a negative.
>>>	If you have encountered this problem or if you know of a 
>>>possible solution, please leave a message here.
>>>Thanks. 
>>>Juan
>> 
>> 
>>G'Day Juan
>> 
>>You have a common problem of not blocking your membrane against
>>non-specific hybridisation.  Nylon membrane needs better blocking than
>>nitrocellulose.  The negative image is due to the probe not hybridising to
>>that part of the membrane and because you have no target = no signal.
>> 
>>For a homologous DNA probe try hybing in: 50% deionised formamide, 2 x PE,
>>7% SDS, 1% BSA (0.5mg/ml Salmon Sperm DNA optional) at 50 degrees.  Wash
>>down to 0.2x SSC 1% SDS at 70.
>> 
>>Good Luck..Klaus
>> 
>>--------------------------------------------------------------------------
>>Catherine Shang
>>Gene Targeting
>>The John Curtin School of Medical Research
>>The Australian National University
>>E-mail: Catherine.Shang at anu.edu.au
>> 
>> 
>>--------------------------------------------------------------------------
> 
> Greetings... Normally I would agree with Catherine.  However, my wife
> and I have experienced this problem in our lab too and it is her impression
> that the problem lies with the lot of N+.  We have always used the standard
> hybridization solution (5X SSC,, 0.2%Ficoll 400,0.2% BSA, 0.2%PVP,0.5%pyro,
> and 1% SDS) ala Maniatis (Salmon sperm optional) and this has worked fine
> for us for years...  until we got this one lot of N+ (#13709) and then all
> h*ll broke loose.  As initially described, our bands were shadowed and the
> background on the blot high.  Only when my wife changed lots of N+ and repeated
> the Southern did it work with low background.  
> 
> Catherine's advise is sound; however, have you had this problem before with
> different lots of N+ or is this a new problem?
> 
> Thanks,
> 
> David
> haviland at kids.wustl.edu


I had the problem of high background in a certain period of time. My colleagues
had the same problem in the same time. I tried very hard to look for the cause.
finally, it worked very well after I made a new batch of buffer and change a 
new tube of 32-P dATP. My feeling was the 32-P (bad batch) and the buffer (too
old).