Is Boehringer Mannheim the only supplier of Dig-labellin

[Joseph Polli]

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> In article <MURIANAP.99.00FE8335 at FOODSCI.PURDUE.EDU>
> MURIANAP at FOODSCI.PURDUE.EDU (Pete Muriana) writes:
>> We are thinking of diverting from biotin-labelling to dig-
labelling nucleic acids.  Is Boehringer the only supplier of dig-
labelling systems?
>> Regards, Peter

>Peter:

>Is there some unsaid reason to switch? I'm using biotin-
labelling. Could you tell me if it would be better to change over 
to dig-labelling?

>*********************************************************
>* Paul N. Hengen, Ph.D. 


Paul,
Actually, we've obtained excellent results with biotin-labelling 
for most of our applications (Southern blots, using PCR to 
incorporate label, biotinylated oligos) except when performing 
**colony lifts**.  We posted this problem a while back to no 
avail; even tried the method recommended by Schleicher & Schuell 
(using proteinase K incubation of filters and wiping with Kim-
wipes) but still got much background which was only "acceptable" 
if using high-copy pBluescript-type vectors but much worse for 
lower copy vectors that we would also like to use and/or 
screening low copy cosmid clones by colony lifts.  My grad. 
student is convinced that dig-labelling (approximately the same 
costs, I believe) will give less background on colony lifts, so 
we're thinking of trying/switching to Dig. ....unless, perhaps, 
someone can convince me otherwise - I'm open to suggestion.

Regards, Peter



Peter and Paul, (but what about Mary- sorry had to start this 
way!)

    I have one suggestion but it depends on what the background 
problems are due to:  1)if the background is all over the filter, 
then my suggestions is to try several lot numbers of filters.  
When we did screening with biotinylated probes, we actually call 
membrane suppliers and requested different lots of filters to 
test.  After testing, we would order the several boxes of filters 
with the lowest background.  This worked well, but to be honest, 
it is kind of a pain.  2) if background is due to bacterial 
colonies, have you tried blocking filters with free biotin, then 
wash, and then block with strepavidin, followed by another wash?  
I hope this helps.

Joe