recombineering for ES cell targeting - retrieval problems (Apr/02/2010 )
I'm trying to generate a construct for ES cell targeting. Firstly, I amplified my construct using PCR and then carried out recombineering into BAC. I checked the obtained colonies with end-sequencing and they are fine. Now I need to retrieve the construct from the BAC together with the adjacent genomic regions which will play a role of homology arms during targeting of ES cells. But this step failed two times already and I don't know what is wrong. I use the following protocol:
1) electroporate pBAD plasmid into the bacteria bearing modified BAC
2) grwowing the bacteria uptaken pBAD at 30 degrees overnight
3) dilute the overnight culture and grow for 2 hours
4) add L-arabinose, incibate 45 minutes at 37
5) electroporate with retrieval vector
6) grow for 3 hours at 37
7) miniprep DNA
8) transform DH5alpha
I have about 9-10 colonies on both induced and uninduced plates
I've had some troubles when I started recombineering... my mistake was that when preparing electrocompetent cells I let the bacteria grow too dense which gave me very few to no colonies.
You could try the SW102 strain instead?