Southern blot problem

lamoreauxw at lamoreauxw at
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

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