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Tansformation fails ,how can i make it ? - (Oct/06/2011 )

hi ,everyone
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 .

-cavalior-

I think in this case you should keep on extracting plasmid from DH5a since you had already got it. It is less than usual but better than nothing from your latest try. It is not surprising that you have problems in amplifying a lentiviral vector since LTRs usually interfere with replication of plasmid in E.coli, maybe due to the recombination? I am not sure though.

-gyma-

gyma on Fri Oct 7 09:21:36 2011 said:


I think in this case you should keep on extracting plasmid from DH5a since you had already got it. It is less than usual but better than nothing from your latest try. It is not surprising that you have problems in amplifying a lentiviral vector since LTRs usually interfere with replication of plasmid in E.coli, maybe due to the recombination? I am not sure though.


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 .

-cavalior-

I have extracted a low-copy plasmid only once so really dont have much experience. In my case, I did 2 midi-prep and got about <1/10 of the amount of high-copy plasmid. I was disappointed to see this result but also felt lucky because I dont need this plasmid much.
I think incubation time doesnt matter much in your case. you may consider trying other strains. here is a link maybe useful for you:
http://www.protocol-online.org/biology-forums-2/posts/22141.html
good luck.

-gyma-