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why I failed generating electrocompetent cells?
Yan Yu
Wednesday, 13 February 2008 17:43 UTC
I have been using a standard protocol to make electrocompetent cells, which is similar to this one: http://openwetware.org/wiki/Knight:Preparing_electrocompetent_cells
I have tried DH10B and XL10 Gold strains for several times, but each time, the competency is very low, much lower than commercial chemical competent cells. I used a 10kb plasmid to test competency. Except for keep aliquots on dry ice before -80 storage (dry ice is not readily available), I think I am pretty good at keeping cells cold. At least that is the best/quickest I can do. I really don’t know what could go wrong. Also, any good commercial electrocompetent cells to recommend for large constructs (~17kb)? Thanks a lot for your help!
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Replies
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Apologies, But I’ll never use an online written protocol untill and unless its a peer revied publised article, Because you don’t have any evidence that the protocol is correct.
Anyways, lets assume the protocol was correct.. so what were the controls?
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Hey Amit, thanks for the reply. This protocol or similar ones can be found everywhere, so it is not just some random protocol. My control is commercial chemical competent cells, DH5alpha subcloning from invitrogen, which showed much higher competency using 2 kinds of plasmid. Any one who have made their own electrocompetent cells?
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