G + C rich sequences

nishir at ohsu.edu nishir at ohsu.edu
Thu Jul 29 11:42:00 EST 1993


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
exact conditions (one person suggested formamide + Denhardts, and another
suggested 1% SDS).  Any other ideas out here?  What's the general opinion about
blocking agents such as salmon sperm DNA and how it should be treated (eg.,
sonication, boiling, concentration)?  Would it help to raise the hyb temp?

Rae
OHSU
Portland OR



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