4 replies to this topic

[PostDocTrauma]

Hi all,

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?

Thanks

[pDNA]

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.

Regards,
p

[PostDocTrauma]

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

[pDNA]

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.

Regards,
p

[PostDocTrauma]

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.