Screening Phage Bank With Degenerate Oligo

Eric Lippay Lippay at vaxd.gat.com
Mon Sep 19 19:54:52 EST 1994


I am screening a Lambda Phage bank with a 17-mer 64 fold degenerate oligo. 
I am having trouble finding a reference that deals with the amount of probe
and specific activity needed to screen with an A-P32 labeled degenerate
oligo.  Currently I am labelling with Terminal Deoxynucleotidyl Transferase
and hybridizing at 2x10 6 cpm/ml for 48 hours.  Is this in the ballpark for
visualizing a positive plaque?  The assay works with a 25-mer that has no
degeneracy, but not with the other.  My wash conditions are 30 min in 6x
SSC, .1 % SDS at 37 C (2x), and 20 min in TMACL at 45 C.  HELP!!!



Eric Lippay
Lippay at vaxd.gat.com



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