Help in Primer designing for expression...

Hi friends..

I am working with pET vector system. for expression of the enzme in
E.coli BL21.
I have to design primer with Nco1 site in forward primer.
so can anybody will suggest me about the placing of ATG site of
ORF...whether it should be in the primer's R.E site or else again i
have to add in the primer aftre the R.E site.

waiting for suggetion from scientific community

Historians believe that in newspost
<[Only registered users see links. ].ne t> on Sun, 19 Jul 2009,
Dr. Hiranya S. Roychowdhury <[Only registered users see links. ]> penned the following
literary masterpiece:

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.

Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.

In <[Only registered users see links. ].com>,
Duncan Clark <[Only registered users see links. ].com> wrote:


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.

AC
--
Email: echo 36434455860060025978157675027927670979097959886449 930P | dc

On Jul 20, 6:43*pm, Aawara Chowdhury <[Only registered users see links. ]>
wrote:

hi friends /....

for calculating the Tm and GC..in primer designing.
whethet i should consider the R.E site sequence along with the primer
sequence..for the calculation or else i should exclude it...

bye take care al.please do respond.