hybridization with heterologous probes

eric anderson e-anderson at ski.mskcc.org
Thu Jun 12 13:33:47 EST 1997


In article <33A0997D.6538 at rug.ac.be>, Van Geldre Els
<Els.VanGeldre at rug.ac.be> wrote:
> 
> Does anyone of you specialists know something about hyubridization 
> conditions with heterologous probes ? Hybridization temperatures, wash 
> conditions,...
> I 'd like to perform "Southern blot" on PCR products obtained with 
> degenerate primes (designed against homologous boxes in already isolated 
> similar enzymes) on genomic DNA in order to identify any "specific PCR 
> product" (for the enzyme I am searching). Any other experiment in order 
> to optimize the PCR and to obtain a "specific PCR product" failed 
> (playing with MgCl2 concentration, annealing temperature, nested PCR). 
> Therefore someone told me to perform this Southern blot with a one of the 
> isolated enzymes as probe. I would like to do the hybridization in Dig 
> Easy Hyb and the manufacturers give conditions applicable if probe and 
> target have 100 % homology (hyb temp 37-42 °C). What is a good temp for 
> me to use ?
> Maybe someone of you has also another suggestion to identify my "specific 
> PCR product" ?
> Thank you for answering,
> Els 
> 

Els,

so as i understand it, you're going to do genomic PCR with degenerate
primers and then probe the resulting smear with a heterologous probe to
try to light up a specific band.

i have 2 suggestions.  the first is to check out page 2.10.11 of Current
Protocols in Mol. Biol.   there's a pretty good discussion of this problem
there.  in a nutshell, a first approximation is that for every 1% decrease
in homology there should be a 1oC reduction in Tm.  the bad news is that
this is a very rough approximation and trial and error is really the only
way to go (but you can use this as a starting point).

the second idea is to try to do some secondary PCR.  if possible, design
an additional pair of degenerate primers inside of the your current pair,
or just use the originals and use them to PCR again using the original PCR
mixture as template.  i have had this work in the past but of course there
are no guarantees.

good luck,

eric

-- 
Eric C. Anderson
Memorial Sloan-Kettering Cancer Center
Sloan-Kettering Institute
1275 York Ave. Box 470
New York, NY  10021
(212) 639-2977
e-anderson at ski.mskcc.org




More information about the Methods mailing list