Ligation of PCR products

jjw at rsbs0.anu.edu.au jjw at rsbs0.anu.edu.au
Mon Jul 22 03:13:50 EST 1991


In article <1991Jul19.125921.14518 at magnus.acs.ohio-state.edu>, gchacko at magnus.acs.ohio-state.edu (George W Chacko) writes:
> In article <9107182217.AA23884 at genbank.bio.net> ST403161 at BROWNVM.BROWN.EDU (Michael Newstein) writes:
>>I have read that it is exceedingly difficult to blunt-end ligate products from
>> PCR  (without cleaving first in internal restriction sites). I am attempting
>>to clone the product of a PCR reaction into a blunt end cutter site (eg SMA 1)
>>in pBluescript. Does anyone have suggestions?
> 
> You could try the TA cloning method. Invitrogen has a kit but it seems fairly
> straighforward. There's a rapid communication in NAR 19:1154 by Marchuk,
> Drumm,Saulino and Collins describing it.
> 
> George

The problem with the Invitrogen kit is the price.  An alternative, suggested
to me by longtime freind Mick Graham late of Calgene (Australia), was to
make your own TA vector by cuting your vector of choice with a Blunt-end 
restriction enzyme (eg SmaI) and then tailing with dideoxyTTP. This 
apparently works well, and it looks a very simple approach which will
allow PCR cloning more efficiently that after blunt end repairing (as it
makes use of the generally A extension put on by the PCR process for base
pairing during the cloning step).  I gather this was written up as a note
for NAR but I don't know if it's out yet.

	cheers,
********************************************************************************

Dr. Jeremy Weinman	Plant Microbe Interaction Group
			Research School of Biological Sciences
			Australian National University

	Email:		jjw at rsbs0.anu.edu.oz
	Phone:		61 6 2495051
	Fax:		61 6 2490754		
	Snail:		PO Box 475, Canberra, ACT 2601, AUSTRALIA

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