E Coli strain for lentiviral packing vector - >10kb vector not transforming (Jul/17/2011 )
I've got some lenti vectors but I can't get the packaging vector (delta R) to transform into my dh5alpha. I only bought the cheapest subcloning efficiency ones (they worked for all the others, including the supposedly toxic insert....
I've tried transforming 2ng, 5ng and 10ng but nothing. I am going to plate the rest of the bacteria but I suspect there won't be much there.
We have some TOP10 in the lab, but can anyone recommend another strain to transform something over 10kb?
you should use a E. coli strain that provides high transformation efficiency and extremely low rates of recombination ...for retroviral stuff people often use STBL2,3 or 4 cells ...as far as i remember the STBL4 are said to be good for large constructs (eg. cosmids).
Maybe you should also increase the amout of DNA you use when working with large plasmids.
I tried to transform with 40ng for both dh5alpha and top10. both didn't work. I can't really afford to spend any money for the next few weeks as it's the end of the financial year.
Guess I will have to buy the STBL lines then
you can also check the quality of your plasmid on an agarose gel ...maybe it is already degraded or broken by repeated freeze-thaw cycles.
I don't have much left so whatever it is will have to do
we don't use ethidium so will have to run at least 50ng.