Nested Deletion paradox

Dave Boyd dboyd at ccu.umanitoba.ca
Tue Apr 19 15:59:52 EST 1994


In article <cmf5-180494123539 at 132.236.156.75>, cmf5 at cornell.edu (Casey M.
Finnerty) wrote:
> 
> Dear Netters:
> 
> I've been sequencing a couple cDNAs using nested deletions (made with the
> Promega Erase-A-Base kit) and have consistently run into something that
> puzzles me.  etc, etc


I've used the Erase-A-Base kit sucessfully 5 times but you are right you
always get unexpected-sized plasmids from each timepoint. However I usually
only screen 3-5 transformants/timepoint and have always gotten at least 1-2
plasmids of the expected size. I'm not sure you could expect better than
that. Your explaination is a good one and could account for some of the
results. I thought the most likely explaination was that the number of
unexpected-sized plasmids would depend on the percentage of supercoiled
plasmid you used to start with; the higher ratio of supercoiled plasmid to
nicked DNA you use the better chance of getting a high percentage of
expected deletions from each timepoint. I always used plasmid DNA that was
purified by MAGIC MINIPREP KITS as starting material.

As for storing your ligations what you describe seems unusual unless your
DNA is getting degraded during storage somehow. Also I've heard that
inactivating the ligase, 65¡C for 10 min., before precipitating the
ligation improves transformation.

dboyd at ccu.umanitoba.ca



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