G + C rich sequences

ottcr at esvx17.es.dupont.com ottcr at esvx17.es.dupont.com
Mon Jul 26 10:59:51 EST 1993


In article <1993Jul23.200713.18270 at ohsu.edu>, nishir at ohsu.edu writes:
>I am trying to work with a 624bp cDNA sequence that is 74% G+C overall and
>there is no smaller stretch that I can cut out that is lower than 70% G + C. 
>We are having a terrible time with background when we try blotting with probes
>made from this sequence.  If fact, even when we do PCR with exact match oligos
>to this sequence we have difficulty amplifying one band (more than a smear of
>others) from cDNA (even with more thermostable polymerases such as Vent and
>going to melting temps of 95-97 deg). We would like to try some in situ
>hybridization but are wary because of the background problems on blots.  I am
>an amateur and do not understand fully the complexity of working with G + C
>rich sequences.  Why is the background hyb (even to filter alone) so  high?
>Should I try a different blocking agent when we prehyb? (we're susing salmon
>sperm DNA). What's the main problem here?  Is it secondary structure?  Is there
>alot of hybridization to repetitive DNA?  Does anyone out there have any
>suggestions on how to deal with G + C rich sequences when doing in situs?
>
>Rae Nishi
>Cell Biolopgy & Anatomy
>OHSU
>Portland OR 

Try doing your prehybridization with 1% sds(+salt etc.)
Then hybridize with 1% sds(+salt etc.)
Add the salmon sperm DNA to the probe before a 2-3 minute boil(denaturing step)
The SDS is a much better agent for bulk blocking than the DNA.
   These conditions described ONLY pertain to membrane blots and will be
different for InSitu applications.
If you have other questions, Please feel free to E-Mail me.
Regards, Charly O.        
E-Mail = ottcr at esvx17.es.dupont.com



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