Problems with making efficient chemically competent cells - (Apr/23/2008 )

Hello,

I am having trouble making efficient chemically competent cells. I am growing DH5alpha cells in LB overnight, diluting them in 500ml of LB and then growing them to an OD reading of 0.5-0.6. From there I spin for 6 minutes, resuspend in 80 ml of cold 0.1M MgCL2, spin for 5 minutes, resuspend in 20 ml of cold 0.1M CaCl2, spin for 5 minutes and resuspend in 3.1 ml of cold 0.1M CaCl2 with 15% glycerol then flash freeze in a dry-ice and ethanol bath. I have also tried to incubate the cells on ice for 10 minutes before the first spin. I keep the cells on ice (or at +4) during the whole experiment but I still seem to get cells with very low transformation efficiencies. Does anyone have any suggestions as to how I should change my protocol to improve my transformation efficiency to around 10^6 (different buffers? different spin times?). Any suggestions will be greatly appreciated. Thank you.

<zloring>

I would recommend Inoue or Hanahan methods for DH5alpha. You can look up in Maniatis for the protocols. I use Inoue method, it's pretty straight forward once you have prepared the transformation buffer as given in the protocol. Oh, and be sure that the bacteria you are going to prepare the stocks, is in good condition. I would recommend to buy a glycerol stock to begin with (commercial bacteria from Invitrogen, Stratagene etc). Every time you need to prepare a new batch, streak a bit of it on a fresh LB-agar plate and grow the culture from that. If you are going to follow the Inoue protocol, it's important to grow the liquid culture at 18-20 degree as given in the protocol.

If you follow the protocol, the transformation eff. should be at least 5x10^6.

Good luck

I would harvest at a lower OD. If your cells start going vegetative, your efficiency will start to suffer.