G + C rich sequences

ottcr at esvx17.es.dupont.com ottcr at esvx17.es.dupont.com
Thu Jul 29 15:46:56 EST 1993


In article <1993Jul29.164200.20218 at ohsu.edu>, nishir at ohsu.edu writes:
>Sorry, i forgot to include the hyb conditions on the genomic Southern that are
>giving us such high background with the G + C-rich probe... we are using 50%
>formamide in 5xSSC + 5x Denhardts + 1% SDS + 10% dextran sulphate +
>250micrograms/ml of salmon sperm DNA at 42 degrees; we wash with 0.1X SSC +
>0.5% SDS at 65 deg.  The suggestions that I've had so far are to use these
  (stuff deleted)
>Rae
>OHSU
>Portland OR

After careful consideration of your comments/questions:
  G&C rich probes generally have a high TM. If they are larger than
oligoes, I would suggest dumping the formamide and increasing the 
temperature to 65 degrees OR 5 degrees < the tm.
  For nylon membranes (& most others) Denhardts and non-homologous
DNA's don't effectively block. 1% SDS, however, should be enough to block
nylon but the actual blocking agent used may not be the problem.
  There are several types/patterns of background on Northerns/Southerns
and they all have different causes. I could use more details about the 
specific bkg. pattern you're observing to guess its origin and eliminate 
the problem.
For further information/discussion call my friend Richard Pfeiff at
1-617-350-9481 OR 1-800-323-8903 (X 9481).

Best Regards, Charly O.

 



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