Rsa digestion.

Justin Hopkins zmga2150 at kcl.ac.uk
Wed Jul 26 07:30:31 EST 2000


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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