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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Though this is too late to help, I should point out that competent cells rarely forgive any variation in freeze and thaw procedure. Bear in mind that you’ve added glycerol to the cells to slow the growth of ice crystals, but a slow freeze could still undo your efforts. The online protocol cited describes a snap freeze in dry ice and ethanol, which would transfer heat out of the tubes very quickly. In theory I suppose someone lacking dry ice could improvise using ethanol chilled to -80 C, with some inconvenience.
To stray slightly from the question, have people had good experience using 200 mM trehalose for competent cell storage? There is even a patent published for storing competent cells lyophilized in the presence of trehalose at room temperature.
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Mike, I think you are right. Later on, I gave up making the competent cells myself. Eventually I realize that the limiting factor is ligation, although super competent cells will help.
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