Hi i need Plasmid containing taq gene
(by news.100.jwgmx At xoxy.net)
Mon Dec 18 07:45:10 EST 2006
Biotechniques once had an article describing the whole process in
detail. Maybe your library carries it ...
One caveat they had: if the Taq is too highly concentrated, your
preparation won't have any apparent polymerase activity, so they
recommended trying a wide dilution range when testing your purified Taq.
NB: It could be "Single-Step Purification of a Thermostable DNA
Polymerase Expressed in Escherichia coli", Urvi J. Desai and Patrick K.
Pfaffle, BioTechniques Vol. 19, No. 5: pp 780-784 (Nov 1995). Not 100%
> 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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