epitope tag: two or one?
chrisb at hgu.mrc.ac.uk
Tue Jun 23 05:07:44 EST 1998
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.
: As an alternative, I would recommend you order two complementary oligos
: encoding ONE tag, with restriction site overlaps at the ends. To introduce
: one tag, anneal the oligos by 5´ 95C, 5´ 70C, and slow cooling to RT, then
: clone into the vector linearized with the chosen enzyme (but NOT
: dephosphorylated). Use a 1000fold molar excess of annealed primers, and you´ll
: end up with over 80% positive clones (i.e. with one copy inserted; inserts
: will be in both possible orientations). Directional cloning is, of course,
: even better.
: For more than one tag, phosphorylate both primers before annealing (or order
: them phosphorylated), then follow the above protocol. Directional cloning is
: almost a must to ensure the oligos are inserted in the wanted orientation
: only. You´ll end up with some clones having 1 copy, but also clones with
: several copies of the insert, encoding several tags. Select one that has the
: number of inserts you want. You´ll need only two relatively short oligos. Very
: cheap, extremely effective.
Something along these lines has been published in:
Nakajima, K. and Yaoita, Y. (1997) `Construction of multiple-epitope
tag sequence by PCR for sensitive western blot analysis.' Nucleic
Acids Res., 25, 2231-2232.
Chris Boyd | from, but not \ MRC Human Genetics Unit,
Christopher.Boyd at hgu.mrc.ac.uk | on behalf of / Western General Hospital,
http://www.hgu.mrc.ac.uk/Users/Christopher.Boyd \ Edinburgh, EH4 2XU, SCOTLAND
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