GJKING at NVRS.AFRC.AC.UK
GJKING at NVRS.AFRC.AC.UK
Thu Jun 17 05:34:00 EST 1993
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
Breeding & Genetics Dept
Horticulture Research International
GJKING at UK.AC.AFRC.NVRS
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