a very unusual problem with ssDNA

Gail Otulakowski gotulak at sickkids.on.ca
Tue Mar 9 14:52:19 EST 1999


Ron Caspi wrote:
> 
> Hi all,
> 
> We have a very unusual problem with ssDNA. Perhaps someone has seen
> that before.
> We prepared a decent amount of ssDNA from pBluescript II (SK minus).
> We used Stratagene's VCS-M13 helper phage, scaled up the Stratagene
> protocol to 50 ml, and got what we consider a good quality ssDNA.
> 
> When we ran the samples on agarose, about 95% of the DNA ran as a
> single band, where you would expect it, 3% were in a faint band
> running a little bit faster (nicked linear DNA?), and a very faint
> band ran where chromosomal DNA would ordinarily be - we suspected that
> was an insignificant contamination with bacterial DNA.
> 
> We aliquoted the DNA, and froze it at -20.
> 
> During the 1st week, we thawed the DNA and everything seemed ok.
> ALAS (you knew that was coming), after about a month, we watched in
> horror as the good band lost about 50% of its brightness, while the
> large band (running as chromosome) gets more and more intensive. It
> seems as if the DNA is converting to a large molecular weight
> aggregate?!
> 
> We thought perhaps we had a chromosomal contamination that dissolves
> slowly in the sample, so we cut it with EcoRI, which would convert the
> large band to a smear. No such luck. The enzyme can't cut any of the
> bands.
> 
> We heat it up at 95 C for 5 minutes, then run it - looks the same.
> 
> We hybridized it to a labeled oligo that should bind only to
> bluescript - and it binds to all bands.
> 
> What is going on? any suggestions?
> 
> Any help is greatly appreciated.
> 
> ________________________________________________________________________
> Ron Caspi
> Center for Molecular Genetics
> University of California San Diego                Phone   (619) 534-2460
> La Jolla  CA  92093-0634                          Fax     (619) 534-7073
Sounds like it is slowly forming an aggregate that is resistant to
restriction enzyme digestion



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