help for ligation

Bob's lab 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
Dong) wrote:

> 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
SDW                                                 5ul

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
other method.



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