Cutting out of a T vector - Cutting sites in primers vs sites in vector (Sep/14/2006 )

Hi there,
If I amplify with primers that introduce cutting sites (say, ClaI and NotI) I should ligate the product into a T vector, to make it easier to cut these sites, right?

But the T vectors (like pGem-T) all have cutting sites including NotI. From memory pGem-T will have a NotI site ~20bp downstream of the insert's NotI.

Is this okay? Can I just do a full digestion (for the maximum time) and hope that every NotI site gets cut? I don't think I'd be able to confirm the digestion on a gel (20bp size difference won't show up).

Is this a common problem, or am I getting worked up over nothing? Last time (and the only time) I ever did TA cloning we weren't so concerned with which site gets cut because it was a promoter. This time I'm making a fusion protein, and we don't want to be frame-shifted!

Cheers,
Eirinn

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No right answer here I am afraid.

Although others will beg to differ, I would forget about the T vector... and clone the PCR product directly into my expression vector (assuming everything is okay and you don't need the T vector to change restriction site... which you shouldn't). If you had design your primers well and included enough overhang bp on the primer ends, there would be no problem for digestion (depending on the quantity, you can give the reaction an overnight digestion).

So in my opinion T vector cloning is a waste of time in this situation, it is an extra step. Why waste time on it.




Yup, 20bp is enogh. Give the NotI an overnight digestion, watch the glycerol which the enzyme comes in. Too much enzyme is a very bad thing. All the NotI site will cut.



I'll say be careful with the junctions... check the fusion strategy on a program like VNTI. That is the easiest way to make sure the strategy will produced an in frame fusion.And do some sequencing work to make sure no mutations were induced into the gene during the PCR.

Best of luck

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Aha, here is the problem. I haven't added any overhang to the primers, because I was worried about Tm. The genes themselves are 70-80% GC (yeah... Chlamydomonas) and the NotI site is 100% GC so the Tm is very high (~80').

Any overhang will increase the Tm, correct? So rather than adding overhang I thought I'd go with TA cloning. What do you think?

Thanks for your help

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PacI! 8bp AT cutter. Not much change to primer Tm.

But seriously, without a modification to the vector, (I assume your vector has no PacI site.) I guess T vector is the easiest route.

Best of luck then.

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