DIG_LABELLING

neale at mbcf.stjude.org neale at mbcf.stjude.org
Fri Jun 18 18:42:13 EST 1993


I'd like to add my experiences of non-isotopic detection. We used BRL's kit
because we had a student in the lab who could not (legally) work with
radioactivity. It worked like a charm for single copy detection on Southern
blot (2ug genomic DNA) the first time with the batch of membrane supplied with
the kit.

Thereafter, it was all she wrote! I had extensive dialogue with BRL at the
inability to reproduce the very first results. We purchased many lots of
replacement membrane from BRL, and they also replaced the membranes on request.
Still, to no avail. The problem was unacceptable background--ie black! 

It works fine for high copy targets (PCR, cloned material at the ng level), but
that's all. We were able to use it for the student's project, but we had
thought to incorporate the procedure into the lab.

A rep from Amersham told me that all the non-isotopic kits are about 10-fold
less sensitive than 32-P, and all vendors are fairly equivalent.

Geoff Neale
St Jude Children's Research Hospital
Elvis Land TN


In article <1993Jun17.093606.13625 at gserv1.dl.ac.uk>, GJKING at NVRS.AFRC.AC.UK writes:
> 
>               Boehringer DIGoxygenin non-radioactive DNA LABELLING
> 
> Our laboratory has had a rich and varied experience with this system over the
> past few years.   We are interested in the experience from other laboratories,
> especially in the effectiveness and consistency of results for single-copy 
> probing in plant genomes (Arabidopsis upwards).
> 
> A few Friday afternoon reflections:
> 
> a. 	When the system was first launched we were able to detect at a high
> sensitivity, exposing the chemiluminescent blots for up to 1 hour with
> reasonable background levels. This was with Brassica genomic DNA probed
> with single/few copy sequences.
> 
> b.	Subsequently we encountered problems with high background, inconsistent
> sensitivity, etc.  Several systematic lines of enquiry were pursued:
> 	i. Change membrane
> 	ii. Batch variation in anti-DIG
> 	iii. Labelling efficiency
> 	iv. Hybridisation buffers
> 	v.  Obtain modified protocols from Boehringer
> 	vi. Use different hybridisation bottles/ovens, etc.
> 	vii. Use different grades of water, etc.
> 
> c.	To cut a very long and extremely expensive story short:
> We have never totally cured the problems of high background. Exposures greater
> than fifteen minutes usually produce black 'autorads'.
> 
>  Boehringer have never satisfied us that they are on top of the situation for
> using the system for routine RFLP blots. They have issued probably 20 
> variations on several themes for the protocol in the last few years - this in
> itself indicates an inherently unstable system.  Any modifications we have 
> made have usually only led to transitory improvements in quality.
> 
> 
> d.	Having said that we have successfully and routinely used the system 
> for detecting high copy number sequences, from blots of PCR products, etc.
> This isn't really pushing the sensitivity of the system, though.
> 
> e.	We are aware that a number of other plant and human labs have 
> experienced these problems over the last few years, although Boehringer
> alledgedly recently claimed that they had not heard of such problems.
> 
> In conclusion, I offer the above comments to warn others that they can waste a 
> LOT of time and money attempting to optimise this system. If anyone out
> there IS routinely using DIG detection for RFLP blots of genomes 0.2 to 2pg
> in size, and reprobing >5 times, it would be useful to start a dialogue.
> 
> It might also be useful to find out how labs have tried and failed to use the
> system. I have had various  hints there are a LOT of dissatisfied customers
> out there who probably think they have done something wrong, when we should
> all be blaming Boehringer !
> 
> We are currently evaluating Amersham's ECL (direct labelling) system for 
> routine screening of RFLP blots. Comments on experiences with this would also
> be appreciated.
> 
> 
> Graham King
> 
> Breeding & Genetics Dept
> Horticulture Research International
> Wellesbourne
> 
> GJKING at UK.AC.AFRC.NVRS
> 



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