jul2 at pitt.edu
Wed Jul 26 03:41:25 EST 2000
Thank you for your help, I think I was not very clear.
The defective allele contains an Rsa site, and heterozygotes are
suppose to have 135bp and 108bp.
But in homozygotes both alleles were defective, so I was expecting to
see just 108 fragments.
It looks like the digestion does not work for homozygotes even partially.
Can you think of any reason for it?
Justin Hopkins wrote:
> > I am genotyping the mice for defective leptin receptor allele (db).
> > According to the protocol I have to run Rsa digestion on the PCR
> > products
> > in order to digest 135 bp fragments into 108 and 25 bp pieces.
> Does this mean that that the defective allele contains an Rsa site,
> absent in the normal allele?
> If so, then maybe the two bands you see in heterozygotes are the 135 and
> 108bp fragments, since the 25bp may be difficult or impossible to see on
> an agarose gel. Then, assuming you mean homozygotes to be for the normal
> allele, you would expect only one band at 135.
> > It seems to work for heterozygotes (I get two bands), but the
> > reaction fails for homozygotes (I get 135 bp band instead of 108 bp).
> > Does anybody have any suggestions?
> > Your help will be appreciated.
> > Julia.
> Hope this may help.
> Justin Hopkins
> Cancer Genetics Laboratory
> 8th Floor, Guy's Tower
> Guy's Hospital
> London SE19RT
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