Rsa digestion.

Justin Hopkins zmga2150 at kcl.ac.uk
Wed Jul 26 12:55:26 EST 2000


The only thing I can think of is to check the sequence to be sure it is the
defective allele that contains the site (or contains a site created by a
miss-match primer - if you are using that method).

What size bands do you obtain from digests for homozygous normal mice?


Julia Liachenko wrote:

> 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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