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

Martin Kennedy mkennedy at chmeds.ac.nz
Sat Mar 19 04:39:35 EST 1994


In article <rcwieg-160394103619 at rcwieg.monsanto.com>, rcwieg at ccmail.monsanto.com (Roger Wiegand) writes:
> In article <2m4kstINNllm at sat.ipp-garching.mpg.de>,
> krasel at alf.biochem.mpg.de (Cornelius Krasel) wrote:
> 
> 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
> Roger

That is interesting, given that the boil preps we make, which don't seem to
work in the ABI but work beautifully for manual sequencing, are always made
from DH5 alpha, an endonuclease-1 minus strain.  I think the proportion of
supercoiled DNA in these preps is probably pretty high.  Maybe the degraded
boiling preps we get from other strains, like TG1, would work in the ABI!!??

-- 
Cheers,

Martin

NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
NN  NN NN           Christchurch School of Medicine            ZZZ
NN   NNNN              Christchurch, New Zealand              ZZZZZZZ
	      Phone (64-3)364-0880  Fax (64-3)364-0750



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