Double insert ligation

David C. Logan david.logan at plant-sciences.oxford.ac.uk
Tue Jan 6 07:47:15 EST 1998


Tim wrote:

>Hi,
>I want to set up a ligation that will joint two DNA fragments together and 
>ligate them into a vector in one reaction. My vector is 7.4kb and will have a 
>3' BamHI end and a 5' SmaI end.  One fragment is only 250bp and has a 5' 
>BamHI end and a 3' KpnI end.  The other fragment is about 2.8kb and has a 5' 
>KpnI end and a 3' SmaI end. i.e I want to goin them tegether like so:

>BamHI.....kpnI................SmaI

>I have pirmers that are specific for both fragments so I can screen my 
>colonies by PCR so I'm not too fussed if I have to screen lots.  I would like 
>to know if anyone has had experience with this sort of thing and what sort of 
>conditions I should use, time and temp. and vector:insert:insert ratio.  Is 
>this a looser from the start?

No it's not a loser. I have done a very similar ligation, with the exception of 
it being completely sticky-ended and consisted of smaller inserts (200bp and 
730bp into a ca. 12kbp vector). So assuming that the larger sizes and inclusion 
of a blunt SmaI doesn't screw things up then I'd go ahead. I used a 1:3:3 
(v:i:i) overnight at 16 degrees C.

Hope this helps, good luck.

David

***************************
david.logan at plants.ox.ac.uk

David C. Logan
Department of Plant Science
University of Oxford
South Parks Road
Oxford
OX1 3RB

Tel: (01865) 275024 (direct)
FAX: (01865) 275074
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