no pellet after centrifugation for making competent cells - (Jul/09/2008 )
I am trying to make electrocompetent cells from bacteria, but after the second wash step with water I don't get any pellet anymore. I already tried to centrifuge at 15000 rpm for 20 minutes but it still looks cloudy. Does someone has advise for me?
Thank you in advance!
Advice: don't use water, luv. Check your protocol: You probably should be using a solution, like cold calcium chloride. The "Protocol Online" methods are very good.
If you want to make electrocompetent cells, you can't use calcium chloride. In the protocols for E. coli, you wash with water, without a solution. But I am using another Enterobacteriaceae and it does not work with water.
Whoops! Sorry about that: you can tell that I make the plain ol' chemically-competent cells, and that I did not read your post correctly. In an attempt to re-deem myself, I looked up a couple of things: first, does your protocol call for any glycerol? Some of the electro-comp protocols look as though they need 10% glycerol in the washes.
Maybe this protocol will help, or give you some direction:
"Recombinant Enterobacter amnigenus highly toxic to Anopheles dirus mosquito larvae." (Curr Microbiol. 2001 Dec;43(6):448-51), Khampang P et al.
Best of luck!
If you have spun at 15000 rpm for 20 minutes, byw, 2 minutes is more than enough, without getting the cell pellet, it is likely the cells are lysed. You definitely don't want to wash electrocompetent cells in any salt solution and it will arc at electroporation! I am not sure if it will work for your enterobacteria, but pasted below is my answer to one of the related post and may worth trying. Good luck!
Here's my way to prepare electrocompetent cells:
1. Grow E. coli in 500ml salt-free LB to OD600=0.8.
2. Spin to pellet the cells and discard the medium.
3. Suspend cells in 500ml ice cold diH2O.
4. Spin to pellet cells again and discard the supernatant.
5. Suspend cells in 2.5ml cold 10% sterile glycerol.
6. Aliquot into 100ul and store at -80*C.
7. Check transformation efficiency and should get 10^9 to 10^10 cfu/ug of pUC19.
* Salt-free LB is the trick. It allows one wash to be sufficient. By avoiding repeated washes, it minimizes cell damage and ensures high transformation efficiency. Of course keep all on ice or 4*C.