Southern problems... too small of a probe?

Ray Lara rlara at nmu.edu
Sat Jun 5 09:58:43 EST 1999


Hello netters...

I am having a problem getting any signal from my genomic Southern hybs.  I
don't get any signal from the digested genomic DNA, however I get get
hybridization and signal of my complete 1 Kb Ladder!  I use high-salt
downward transfer, which gives me complete transfer in 5 hours, and then I
hybridize.  I am using the BH DIG Detection system. 

I am hybridizing with a 116bp DIG PCR synthesized probe at ~100ng per ml of
hyb solution and 0.25mg/ml herring sperm DNA.  I hybridize overnight at 64
degrees C then wash:

2 x 5 minutes in 2X SSC, 0.1%SDS at room temperature; then 
2 x 30 min 0.5X SSC, 0.1% SDS at 55 degrees C.  
  
What am I doing wrong?  I use a LOT of target genomic DNA (40ug!), and a
lot more probe (100ng/ml) then BH recommends (25ng/ml), and I am getting no
background problems...  

If anyone using the BH DIG system can help, please e-mail with comments or
suggestions
Thanx to all in advance.

Jonathan



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