Double insert ligation

[Victor Levenson]

BMBTJY at leeds.ac.uk (T.J. Young) 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?
>Thanks in advance.
>Tim ;-) 


Hi, Tim:

No, that's not a looser. It is better if all ends are different and
sticky, but you have a  fair chance of getting this done with one
blunt end as well. When I did a similar thing I used same molar
amounts of all three fragments, rt overnight and got a fair number of
correst recombinants. The key issue is incompatibility of ends for any
combination of fragments other than desired.


Good luck,

Victor


Victor Levenson MD/PhD
Res Asst Prof
UIC, Dept of Genetics M/C 669
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