epitope tag: two or one?
Frank O. Fackelmayer
fof1 at chclu.chemie.uni-konstanz.de
Tue Jun 23 10:50:00 EST 1998
Chris Boyd wrote:
> Frank O. Fackelmayer (fof1 at chclu.chemie.uni-konstanz.de) wrote:
> : Chris Boyd wrote:
> : > : Chris Boyd and Darren Natale: It is cheap to add one epitope but it will be
> : > : more difficult to add multiple epitopes by PCR alone.
> : >
> : > Why? You can add 4 HA tags to the C-terminus in one go with a primer
> : > of only (eg) 20 [for initial priming] + 4*27 [the tags] + "TGAATTC"
> : > [stop codon + EcoRI site] + 1 [for luck] = 132 nt: even long oligos
> : > are not expensive these days. You can eliminate the possibility of
> : > mispriming by creative choice of wobble position bases in the tags.
> : > Admittedly, after PCR you MUST sequence to verify the constructs, but I
> : > still think it's more cost-effective than using tagging vectors (except
> : > perhaps for one-offs).
> : I wouldn´t trust oligos of more than 100nt. The chemistry of oligo synthesis
> : is at its limits there, no matter how good the chemist is who makes the oligo.
> They're still usable for PCR though, as many published papers testify.
Yes, they can be used for PCR, but that does not say they have the correct
sequence. Have some 10 correct bases at the 3´ end, and viola, your PCR will
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