Help in Primer designing for expression...
(by aawara from pontiff-playground.org)
Mon Jul 20 08:43:01 EST 2009
In <DAwNpmA59GZKFArl from abuse.plus.com>,
Duncan Clark <blackhole from abuse.plus.com> wrote:
> Historians believe that in newspost
><mailman.618.1248049891.21502.methods from net.bio.net> on Sun, 19 Jul 2009,
> Dr. Hiranya S. Roychowdhury <hroychow from nmsu.edu> penned the following
> literary masterpiece:
>>Since the ATG start codon is part of the NCO1 sequence, we use that RE
>>site for in-frame ligation. So, if your amplicon is digested and
>>religated at the NCO1 site, you should be fine.
> Just remember that for Nco I, the codon following the ATG must start
> with a G either original or you must add one i.e. CC - ATG - GNN
> Also pick the correct pET21 i.e. pET21d :-)
> For this reason I much prefer to use Nde I whenever possible i.e.
> CATATG. No problems with extra aa's.
NcoI can be fairly versatile as well. An NcoI overhang is compatible
with BspHI (TCATGA) and PciI (ACATGT).
So, the second codon (after ATG) can begin with G (NcoI), A(BspHI), or
T(PciI). Between these three, the second amino-acid can be anything
other than histidine and proline.
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