epitope tag: two or one?

Darren A. Natale dnatale at box-d.nih.gov.nospam
Wed Jun 24 07:49:39 EST 1998


Guy Oshiro originally wrote:

> It is commone to repeat the epitope tag.  It is common to see 3xHA tags
> and 9xMyc tags in the literture and in my clones.  You get a much
> better signal with the repeats than the single.

To which I asked:

> Okay, so does anybody have vectors for these multiple tags?  I (and
> likely others) would be interested in vectors that allow for N-terminal
> or C-terminal tags of hexahis, FLAG, c-myc, HA, and any other you might
> have.

Which caused a "life on the edge" vs "wouldn't be prudent" exchange of
viewpoints regarding the use of oligonucleotides to make the multiple
tags.
Chris Boyd suggested a single >100 nt oligo could do it, while Frank O.
Fackelmayer thought it better to use multiple small (no greater than 50
nt)
double stranded adaptor-type oligos followed by cloning.  (I presume the
multiple-tag clone would be engineered with an restriction site for
subsequent
cloning of the coging region of interest.)

Unfortunately, no one responded to my request for a vector of the sort I 
described.  Such a vector would need to be made in the way described by
Dr. Fackelmayer, with a restriction site engineered at either the 5' or
3' end
of the tag for cloning of a cDNA of interest, which itself would likely
need
to be made by PCR to engineer the restriction site in a suitable frame
and
location.

For the record, prior to my request, I had already started to make one
such
vector (still in progress), by the multiple adaptor method, whereby I
used
oligos (~35 nts) with 5' phosphates (for those that would be internal
(i.e.,
ligated to another oligo) and designed to have large overhangs that
would 
complement other oligos.  Of the 12 clones I picked, 11 seem to have the
multitag insert.  Sequencing will tell the final tale.

Note that I am making only one of many possible vectors (one type of
tag, for 
use at one end).  Now, surely someone else has done this!  Speak up!
Speak up!

Thanks for listening :)

D. Natale
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