LR Reaction - Unwanted Homologous Recombinations
Posted 04 October 2010 - 06:33 AM
Posted 22 October 2010 - 04:03 PM
I resolve it by:
1. using Stbl3 cells (originally) and HB101 (from Zymo research).
2. You should use only LB media (on all steps, including overgrow cells after transformation and agar plates), SOC is not good.
3. cultivate cells (plates and for mini/maxi preps) at 25-28C.
Make a search in google. I have read paper where was shown recombination of DNA in cells when they cultivated with temperature more than 30C.
PS. for LR I am using 150ng dest vector, 100 ng entry clones. I am doing usually dest + 3 entry clones (3 different inserts).
Edited by maxmv, 22 October 2010 - 04:07 PM.