Hi i need Plasmid containing taq gene
Jose de las Heras
(by josenet At tiscali.co.uk)
Mon Dec 18 04:34:28 EST 2006
In our lab we have prepared our own Taq, in a much cruder way, but it works.
Essentially it comes down to making a protein extract froma bug that
contains an expression vector with the Taq in it... after induction you
produce loads of it, as seen on a gel simply by Coomasie staining. We
inactivated most things by heating (Taq is supposed to be resistant, right?)
and just compare how the extract works against a commercial Taq, to assess
how many units per ul we have. It's been a long time since I last did that,
but it worked and we got enough Taq to last us several years...
"WS" <novalidaddress At nurfuerspam.de> wrote in message
news:1166359198.227604.305640 At l12g2000cwl.googlegroups.com...
> 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
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