...Or do the slightly-less-new-but-not-quite-old school technique:
QuickChange. Find a vector with roughly the properties you want, and just
quickchange
mutagenize in NdeI and ClaI sites at the desired positions. Works like a
charm
for me, assuming the vector is <10Kb or so, and I typically buy the various
components from the cheapest suppliers rather than the er... *cough*
$$$tratagene kit.*
Or, if you go the QuickChange route, might as well do the entire cloning
using it!
We now routinely clone in the whole genes using it: PCR your gene with
primers that anneal to the plasmid, use PCR product in place of QuickChange
primers. That's all. Works like a charm. I was very excited when I invented
it couple years ago - only to discover that I invented a wheel - the
technique (with few variations) has been published three times already (and
no one paper cited another; can find refs. and give my protocol if
interested).
This way, just like with SLIC, you can put anything into any position on
the plasmid but, IMHO, with much less fuss - no fiddling with vector
besides miniprep and the only enzymes used throughout are polymerase and DpnI.
Dima
