Double insert ligation

T.J. Young BMBTJY at leeds.ac.uk
Tue Jan 6 05:17:39 EST 1998


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?
Thanks in advance.
Tim ;-) 



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