Subcloning PCR products

[John Watson]

> > Joy Richman wrote:
> >
> > Dear Netters,
> >
> > I would like to subclone some PCR products as quickly as possible. I am
> > prepared to spend money on a kit but would like some reviews on the kits
> > that are out there. Also a comparison of the blunt ended, TA cloning and
> > cloning using specific primers provided with the kit would be useful.

I'd have to say that it depends on the specifics of your experiment.  If you are 
using Taq polymerase (or one of those two-enzyme mixtures), then cloning into a 
T-vector can be an efficient way to go, provided of course you don't mind 
sequencing both strands.  I have very good results over the last 18 months with 
Invitrogen's T-vector kit, altough I use homemade electro-competent cells rather 
than the competent cells provided in the kit (I save those for more routine 
subcloning operations).  If you are using a proofreading polymerase then you can't 
use T-vectors, and in these cases I do prefer adding restriction sites, digesting 
and directional cloning into the vector of my choice.  I haven't tried the various 
blunt-end PCR cloning kits out there (although I do have Invitrogen's PCR-Blunt in 
the freezer), although the lab down the hall has had good success with a 5'->3' kit 
(I forget the name).

Good luck,

AJW
----------------
John Watson
Bristol-Myers Squibb Co.
watson_j at bms.com
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