help for ligation
pdxsmi at pdn1.gene.nottingham.ac.uk
Tue Sep 24 11:29:19 EST 1996
In article <51upc9$335 at news.iastate.edu>, qfdong at iastate.edu (Qunfeng
> Hi, sorry if this topic has been discussed before. One of my friend asked me
> what's best ratio between insert and vector. His insert is about 900 bps and
> his vector is about 12 kb, Both are cut by EcorI. Anyone experienced with
> large vector ligation before? and what's the DNA amount in ligation?
> Thanks a lot.
> Qunfeng Dong
> qfdong at iastate.edu
1. Proportion of insert to vector should be adjusted with regard to the
respective DNA concentrations.
There should be an excess of insert to vector of around 5 to 1.
2. Set up ligations in a 0.5 ml Eppendorf tube -
Digested insert DNA, eg 1ul
Digested vector DNA , eg 5ul
5x T4 DNA ligase reaction buffer 3ul
T4 DNA ligase 1ul
3. Incubation is 15 degrees C for 4 to 12 hours, normally 4 hours.
(4. Add 5ul directly to approximately 200ul competent cells).
WORKS everytime for me! To increase ligation efficiency you could purify
the appropriate digested fragments (vector and insert) say by Geneclean or
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