Southern blot problem

lamoreauxw at msuvx1.memst.edu lamoreauxw at msuvx1.memst.edu
Tue Mar 15 09:59:49 EST 1994


I am having a problem with a Southern assay that I could use some input with.
The organism is the American dog tick.  The target is the vitellogenin gene
(yolk protein).  I have cut 10 micrograms of total genomic DNA with several REs
and probed with a biotinylated 45-mer oligonucelotide and used the PhotoGene
detection system (BRL).  I know the reagents work and that the probe is
biotinylated.  After running an overnight (18 hr) hybridization and washing at
50oC, I get no signal.

Question 1: Is 50oC too hot, resulting in melting bonds?

Question 2: Is 10 micrograms too little? Should I up the concentration to 30
micrograms, or more?

Question 3: Should 32P be used instead of biotinylation?

Thanks in advance

Bill Lamoreaux
The University of Memphis
Department of Biology
Division of Molecular Sciences and Microbiology
Memphis TN 38152
LAMOREAUXW at MEMSTVX1.MEMST.EDU



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