Rsa digestion.

Justin Hopkins zmga2150 at kcl.ac.uk
Thu Jul 27 05:04:30 EST 2000



> The sequence is write because the enzyme partially digests heterozygotes.

This doesn't show which allele is being digested.

>
> If you try to digest homozygous wild type mice, you will get one 135 bp band.

Have you done this?

>
> I am trying to think of conditions  that can prevent restrictiong enzyme from
> working.
> Do you know any?
>

Sounds like your enzyme is working fine, the digestion of heterozygotes controls
for this. If your template DNA was prepared the same way for all mice, and the
same PCR master mix used in each reaction, the only condition that will inhibit
the restriction enzyme is absence of its target sequence! I suggest you take a
few steps back to try and explain this rather than blaming the poor enzyme!

>
> 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

--
Justin Hopkins
Cancer Genetics Laboratory
8th Floor, Guy's Tower
Guy's Hospital
London SE19RT
Ph  +44 207 955 5000 ext 5581
Fax +44 207 955 8762







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