His-tag on primer?

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wetheril
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His-tag on primer?

Post by wetheril » Tue Dec 12, 2006 12:26 am

Hi, I ventured into this forum because this is the first semester I started to take biotechnology classes, and this place seems like the perfect place to find some answers to questions.

I want to design a primer for PCR that will create DNA that encodes for a his-tagged protein (the DNA will be transfected into bacteria), where should the his tag be located relative to the coding sequence on the primer?

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LilKim
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Post by LilKim » Tue Dec 12, 2006 5:35 am

so what you're trying to do is greate a protien containing a HIS tag?

First of all you're getting the definition of a primer confused.

Check these links:
http://en.wikipedia.org/wiki/Polymerase_Chain_Reaction

and (a video)

http://www.maxanim.com/genetics/PCR/PCR.htm

So essentially you'll need to do some cloning to get your gene of interest in a vector in frame with the coding sequence of the HIS tag. I'm not sure where you should put the his TAG (in the 5 or 3 prime end of the coding region). You'd probably have to figure this out by trial and error or ask someone who knows about the particular protein you're trying to label.

After you've finished your cloning you can transform your bacteria.

please let me know if you need further direction or clarification.

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canalon
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Post by canalon » Tue Dec 12, 2006 4:24 pm

LilKim is not completely correct, even if I think her method is way safer than the insertion of a His-Tag by PCR it could be done. Basically you have to insert an overhang coding for 6His in any end of your gene (+ ~20bp for the annealing) and amplify your gene. But if that solves the frame problem, this creates other : you also have to insert a start or stop codon in your overhang, this can create non specific hybridization in your PCR etc...
Patrick

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oppox
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Post by oppox » Wed Dec 13, 2006 1:11 am

you might have too incorporate a trombin site to if u want to be able to cut the his tag away. I have thought of this too when I had problems, why do u want the his-tag on your primers, is it just an idea?

sdekivit
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Post by sdekivit » Fri Dec 15, 2006 5:04 pm

you can synthesize a primer coding for 6 His-residues, a protease cleavage site and a stop codon during the PCR reaction added to the reverse primer.

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