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

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

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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