4 fragment ligations.....?

Ned Mantei mantei at cell.biol.ethz.ch
Wed Aug 23 01:17:29 EST 2000


In article <220820001600352406%jpcd100 at mole.bio.cam.ac.uk>, 
jpcd100 at mole.bio.cam.ac.uk wrote:


>How important is the molar ratio of the various frags, Ned? 

I use 5 fmol of vector and 10 fmol of each insert fragment (including 
double-stranded oligos--some people seem to use way too much) in a 10 ul 
ligase reaction. I estimate concentrations by running a bit of each 
fragment on an agarose gel and comparing the staining to a standard.

Optimal concentrations for 2-fragment ligations are discussed in Table 
3, "A practical set of recommendations for ligation", in Revie, et al., 
Nucleic Acids Res 16:10301-10321, 1988, "Kinetic analysis for 
optimization of DNA ligation reactions". Worth reading the whole paper 
carefully.

As to the "chain reaction cloning", I don't know.

-- 
Ned Mantei
Department of Cell Biology, Swiss Federal Institute of Technology
CH-8093 Zurich, Switzerland






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