PCR Cloning

Dom Spinella dspinella at chugaibio.com
Wed Jul 23 16:46:16 EST 1997


Dr. Philip M Sass writes:

> I am trying to clone a membrane associated protein. We have purified
> the protein (human) and managed to obtain 18 amino acids from the
> amino-terminus. The sequence is KPDEEDHVLVLVKGNFDV. Can anyone help us
> to design oligos for PCR cloning? Any suggestions about PCR conditions?
> 
> thanks,

Philip:

I took the liberty of running a BLAST search on your sequence against
Swisprot, and it sure looks a lot like its derived from
protein-disulfide isomerase (1 amino acid off).  I don't know anything
about this enzyme, but I can pretty much bet that if your sequence isn't
in fact derived from PDI, any degenerate PCR primers you design are
likely to amplify up the protein-disulfide isomerase gene.  You might
take a look at the sequence before you attempt primer design.  Good
luck  -- D.G. Spinella



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