Subcloing PCR Products

[jms93]

In article <Ds7ww6.7q7 at uns.bris.ac.uk>, bijgh at zeus.bris.ac.uk says...
>
>Minsoo Yoon (ms.yoon at auckland.ac.nz) wrote:
>
>: Why don't you try T-A cloning?
>: You can either buy (from Promega or Invitro gene) or make your own t 
vector.
>: I've been using t vector to clone my PCR products. It's much easier 
and
>: more efficient than traditional blunt-end ligation.
>
>I'd have to agree with this and add that once you have your PCR safely 
in 
>the TA plasmid you can amplify up as much as you want, and when it comes 
>to restriction, you *know* that you have cut at both sites. 
>
>Jared
>
>--
>Jared Head     at the Department of Biochemistry, University of Bristol
>
>    "If anybody wants to clap," said Eeyore when he had read this, 
>                       "now is the time to do it."


I would agree with this too! PCR with non-proofreading enzymes such as 
Taq, Tfl etc., produce A overhangs which facilitate it's sub-cloning into 
a vector with T overhangs. We use the pGEM-T vector from Promega. First 
we band prep/purify our DNA products before sub-cloning our procedures 
which is a very wise thing to do. This way you are sure it is the band(s) 
of interest that you are sub-cloning. Routinely, we band purify 40 
microlitres of a standard PCR reaction and get good yield back. We 
estimate the concentration of the DNA simply by band intensity on an 
ethidium bromide gel and then use the appropriate amount in the ligation 
reaction. 

Haapy cloning!

Jonathan Shillingford
jms93 at aber.ac.uk