Double insert ligation
BMBTJY at leeds.ac.uk
Tue Jan 6 05:17:39 EST 1998
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:
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.
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