James N Petitte
jnppo at unity.ncsu.edu
Thu Jun 17 10:51:17 EST 1993
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
: 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.
jnppo at unity.ncsu.edu
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