Please help! Can Dh5alpha Competent cells be used for electroporation? - (Jun/20/2006 )
I need to clone a gene and I have DH5 alpha cells, I thought they could be used for electroporation, but when I got the box it says chemically competent. Does that means I can not electroporate ???
Thanks for any help!!!
You can grow up a cell culture to OD 0.5 from your chemically competent cells, wash 2x in ice cold DI water, and resuspend in 10% volume of original culture of DI water + 15% glycerol (ice cold) aliquot (keeping cold) and freeze at -80. Those cells should then be fine for electroporation.
agree with phage434.
The prepration of competent cells is different for chemically cometent or eletrical competent.
Thus, a chemical cell preparation contains too much salts for electroporation, resulting in an electrical arc during electrical shock.
The protocol of phage allows you to obtain quick electrocometent cells.
is there a reason you have to electroporate? if you are ready to transform now, why spend the time growing up and washing new cells, when you can heat-shock transform them? unless they are quite old and not likely to transform with good efficiency?
I've cloned using chemically competent cells in the previous lab I was, because we didn't have an electroporator. And we were even making the cells ourselves (that means lower transformation efficiency than the one you buy). Give it a try and if id doesn't work, you can always use more cells and more DNA. And if you make the electrocompetent cells yourselves, you cannot be sure they are more competent than the ones you have: ones I had a very bad efficiency!
Electrocompetent cells are supposed to be more efficient than chemically competent cells. But if u buy from the companies, they have similar efficiencies. So why wait for until u make electrocompetent cells, go ahead and heat shock it like aimikins suggested.
Thanks all of you for the help. It was of much help!!! It is so true, why not doing heat shock if my cells are already made for it? But it is good to know I can make them electrocompetent in the future.
You are all awsome!!!!! That's why I love this site so much.
I use both chemically competent and electrocompetent cells.
You shouldn't have an issue with the chemically competent cells if you follow the directions.
I've just spent some time working on making good chemically competent cells, so if people want ones as good as you can buy, check out this page: