hybridization problems

Klaus.Matthaei at anu.edu.au Klaus.Matthaei at anu.edu.au
Mon Oct 5 23:01:42 EST 1992


>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

Dear David

This is Klaus.  Firstly I must point out that Bill Warren is correct, I
have not had sex change rather Catherine my technician (and prized right
hand) was using my E-mail and altered the automatic signature which I
failed to check.  My suggestion to the network therefore had her signature.
 This just goes to show that if you do things automatically this can
sometimes automatically get you into trouble.  Sorry about that.

But back to hybridisations.  You are correct different batches of
positively charged nylon membrane can have variations in the ammount of
charge on them and give huge differences not only in the signal obtained
but also the amount of background that you get because of the blocking
required.  Many years ago we were involved in developing the method of
alkaline transfer for southerns (Reed and Mann NAR 1986) and the membrane
(Biorad, Zetaprobe) that we were using worked extremely well.  A little
later however the manufacturer changed the amount of charge on the
membranes and we ended up with poor signal and massive background.  After
much hassle we eventually identified conditions to overcome this problem
which includes the 7% SDS that I mentioned.  This reduces background but
also increases the signal about 4 fold.  Under these conditions I have
never had a problem with Hybond N+ but using the Maniatis conditions you
may still see variability, that I don't know.   But certainly different
brands of membranes and sometimes batches of the same membrane will behave
differently using some methods.

I hope that this helps.

Cheers, Klaus.
--------------------------------------------------------------------------
Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

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