a very unusual problem with ssDNA

Malay curiouser at ccmb.ap.nic.in
Mon Mar 8 03:13:59 EST 1999



->
>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.
>


Did you try running a denaturing gel? How big your probe is?

Malay

Malay Kumar Basu
Centre for Cellular and Molecular Biology
Hyderabad 500007
I N D I A

Fax: (00-91)40-7171195
Phone: (00-91)40-7172241
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curiouser at ccmb.ap.nic.in






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