Rsa digestion.

Julia Liachenko jul2 at pitt.edu
Wed Jul 26 07:49:28 EST 2000


The sequence is write because the enzyme partially digests heterozygotes.
If you try to digest homozygous wild type mice, you will get one 135 bp band.
I am trying to think of conditions  that can prevent restrictiong enzyme from
working.
Do you know any?



Justin Hopkins wrote:

> 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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