PCR Subcloning with restriction sites

Susanne Rohrer srohrer at immv.unizh.ch
Mon Mar 27 11:27:12 EST 2000



Jan-Henner Wurmbach wrote:

> ...PCR to amplify from the pGEM-T template, but with
> > modified primers that included BamHI on the 5' end of the forward primer
> > and XhoI on the 5' end of the reverse primer and clone again into
> > pGEM-T. This presumably would give us these restiction sites and allow
> > easy subcloning into the pET vector in-frame.

I agree with Jan that it's convenient enough to clone directly into pET.
XhoI is a bad cutter - even with 3 bp overhang and overnight digestion, only
75% is cut.  I've  used it , but I only got about 3 clones  - my other enzyme
was NdeI, just as bad. Bam is OK, though.

> However, after doing the
> > PCR and cloning back into pGEM-T Easy, we found that BamHI nor XhoI
> > would cut the plasmid, although the pGEM-T Easy EcoRI sites adjacent to
> > the cloning site digested fine. Our enzymes are good, so I'm not sure
> > what could account for these difficulties.
>
> I am right now doing something similar (with pET26b).
> Ok, you tested your Enzyme.
> Your PCR works, OK. (Sequenced Product? You want to Express that!)

I'd recommend to sequence the very clone you will be expressing from, so
you're sure you haven't subcloned a mutation.

>
> Why don´t you just cut the PCR-product with BamHI and XhoI, show the
> shortening of the Procuct on gel (if lenght of Product allows),

That should be very hard, I think. but after an overnight digestion, you can
be sure the digestion is as complete as can be - subtracting the 25% from
Xho...  By the way I'd reduce the amount of enzyme to prevent star activity.


Susanne





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