Rsa digestion.

Julia Liachenko 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?
Regards, Julia.


Justin Hopkins wrote:

> Hi
>
> >
> > 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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