blunt-end ligation

Susanne Rohrer "srohrer(removethis)" at immv.unizh.ch
Fri Oct 27 02:24:08 EST 2000


MBI's T4 ligase protocol has the following modifications, and that worked
too, but it seems they might be unnecessary:

"Dr. Hiranya S. Roychowdhury" wrote:

> 5ng vector (dephosphorylated)
> 5ng insert

3-10 x more insert than vector

>
> 2uL of the 5x ligation buffer
> 1 U ligase

4U

>
> total vol. to 10uL

add 1/10 vol 50% PEG4000 (endconc. 5%)

>
> 30 min to overnight at RT

2h to overnight at 16°C

> Transform comp. cells (100uL) with 5uL of the rxn.
> Recover at 37 C w/ very gentle shaking for 30-45min.
> Plate.

Susanne






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