DIG_LABELLING

[James N Petitte]

GJKING at NVRS.AFRC.AC.UK wrote:

:               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 !

Thanks for the post, I thought it was our technique but we have
experienced the same problems.  We can use it to detect tandem repeats in
dot-blots, otherwise (viz. single gene detection) it is worthless.  We have
also tried the Bio-Rad system and found that extremely time consuming to
generate probes.


: 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.

Keep us posted on this.

Thanks

James Petitte
NCSU
jnppo at unity.ncsu.edu