A GOOD KIT FOR PCR CLONING

d.a.bailey gen124 at nof.abdn.ac.uk
Wed Feb 1 12:26:14 EST 1995


: > 
: > >  I am looking for a good kit for PCR cloning. Can you give me some ideas 
: > > , based on your EXPERENCE?
: > 
: > The best so far for me is the TA cloning kit from Invitrogen.  With a 25
: > microliter PCR I use 2 to 4 microliters directly in 25 microliters of
: > competent cells. Follow their transformation protocol, plate the entire
: > transformation on two plates and voila!- Bunches of white colonies and a
: > few blue ones.

: Er.  Maybe I'm missing something here.  Don't you need a ligation in there
: somewhere between the PCR and the transformation?  If not, I'd be very
: interested to hear the protocol!

I recently had a go with the Promega T-vector system which worked pretty
well, roughly 50/50 whites to blues. Although only about half of the whites
contained an insert.

	I sequenced a couple of the whites with no insert and found deletions
in the polylinker which is presumably due to the a bit of residual
exonuclease activity in the ligase (yes this protocol does involve a ligation
step ;-)

	The kit comes with 50ul of pre-cut vector, a test insert and ligase.
I think you can also buy in the comp. cells if you have more money than
sense.

	I have used it it clone fragments from 0.4 to 3kb following the
standard protocol and it's worked ok, hope this is of use to you.

--

______________________________________________________________________
bye bye!      | David A. Bailey,            |
              | Molecular and Cell Biology, |  
        0     | Marischal College,          |  "..root toot tooty,
       /O\/   | University of Aberdeen.     |    Oh what a beauty..!"
        \\    | Tel. (0224) 273188.         |
              | d.a.bailey at abdn.ac.uk       |
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