Posted 06 October 2011 - 09:28 PM
i was amplifying a lentiviral vector ,10.4 kb .During the first weeks ,i used DH5a competent cell and heatshock for transformation，but little plasmid DNA could be extracted.
Then ,i learned that the vectou contains low copy replicon,F1 . So ,i planned to use STBL2\3 and 2YT medium instead. Here problems come: The same transformation method, the same amount of plasmid , but no clones could be found on the LB plates . At the same time , the contral LB plate mediated by DH5a turns out very well .
How can i do ?
Thanks a lot .
Posted 07 October 2011 - 01:21 AM
Posted 07 October 2011 - 09:06 AM
Thanks for your reply ,gyma.
I have been trying for many times , but the concentration was never greater than 0.2 ug/ul . So i have to transfor the vector to STBL2/3 in the hope that more plasmid could be extracted.
I am wondering whether it wil work to prolong incubation time .
Posted 07 October 2011 - 06:39 PM
I think incubation time doesnt matter much in your case. you may consider trying other strains. here is a link maybe useful for you: