Hi i need Plasmid containing taq gene

[WS]

Dear Imran,

Just had a discussion with a colleague regarding making your own Taq. I
developed a short concept on how to do it, I would like to share it
here. However, I'm sure its quite crude yet and won't work in that way.
However, I believe it's a good point to start on how to *really* do it.
So you all here are invited to dissect this cooking recipe and to
improve it and to add your own thoughts!

*******
Dear ...

I never did that nor do I exactly know how to do it. This is, because
Taq is so horribly cheap here that it does not make any sense to take
the risk of not knowing how pure and how active the enzyme is, not
talking at all about possible DNA contamination that might produce
wrong results.

First, I'd look how other people did it, eg. at http://www.pubmed.org
and http://www.freepatentsonline.com and try to gather as much
information as possible. That might be a very good students exercise
for third years or master students as well.

Basically, I'd add a His-tag or something alike to the C-terminus to
facilitate the purification. Then choose a protease deficient strain
(i.e. a strain dedicated for protein production) as HB2151 (a
comprehensive list is in your NEB catalog or may be found at
http://www.neb.com, they might give away some strains for free when
your order some restriction enzymes).
Then fractionate your bacterial lysate by ammonium sulfate
precipitation with stepwise increasing concentrations and purify by
affinity resin according to your tag. (alternatively, if you don't want
a tag etc, you might perform traditional ion exchange chromatography)
Then add DNAse to remove any residual DNA contamination (you might
check with some E coli primers and Taq to be known to work), then heat
to the boil for some time to kill DNAse an any other contaminating
protein. Check purity by SDS-PAGE, you should have a single band. Put
in storage buffer a you can read in any commercial Taq data sheet and
store in aliquots at -20.

Written down in 5 minutes that should keep you busy for at least 8
weeks.

Enjoy!

Wolfgang