Alas, poor "magic"! I knew thee well.

Roger Wiegand rcwieg at ccmail.monsanto.com
Wed Mar 16 11:36:19 EST 1994


In article <2m4kstINNllm at sat.ipp-garching.mpg.de>,
krasel at alf.biochem.mpg.de (Cornelius Krasel) wrote:


> 
> It seems that automatic sequencing on ABI machines with Taq polymerase is
> more sensitive to contamination than manual sequencing with Sequenase.
> A guy in our lab brought his DNA and primers to the sequence service
> (who use an ABI sequencer) and had absolutely clean lanes, no band
> whatsoever; then he used the same DNA (which was, BTW, Quiagen-purified)
> with a Sequenase kit from USB, and was lucky. The people who operate
> the ABI machine have told us that this occurs from time to time. Has
> anyone (except Martin, see above) had similar experiences?
> 
> --Cornelius.
> 

We've had a rash of DNA being "too good" for cycle sequencing using the ABI
kit. DNA will give absolutely no signal unless it is either boiled for 10
min or nicked with an enzyme, them voila, beautiful sequence. I've also
documented this for PCR--preps that have a very high percentage of
supercoiled molecules don't work well or at all. There may also be a strain
dependence related to supercoiling. I'm beginning to suspect that in these
procedures you're only looking at the nicked fraction of molecules with any
efficiency

-- 
Thanks,
Roger

rcwieg at ccmail.monsanto.com



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