Double insert ligation

Ned Mantei mantei at neuro.biol.ethz.ch
Tue Jan 6 07:07:13 EST 1998


In article <68t0co$8e0_001 at leeds.ac.uk>, 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

There is absolutely no problem with this, and such things will work even
with 4 insert fragments plus vector. Use about 5 femtomols vector and 10
femtomols of each fragment in 10 ul. If you want to suppress background
even more than it would be anyway by virtue of the two different ends,
treat the vector with phosphatase (and get rid of the phosphatase
completely by phenol extraction and ethanol preciptiation). 90% or so of
the colonies should contain the right plasmids--there is no need to screen
lots of colonies.

-- 
Ned Mantei
Dept. of Neurobiology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland
mantei at neuro.biol.ethz.ch   Fax: +41-1-633-1046



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