IUBio

a very unusual problem with ssDNA

Ron Caspi rcaspi at popmail.ucsd.edu
Fri Mar 5 19:16:57 EST 1999


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



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