Propagating ccdB survival cells - (Dec/06/2010 )

Hi! I am wanting to know if anyone has had success propagating and creating competant ccdB survival cells from Invitrogen. These are the ones necessary for propagating destination vectors for gateway cloning. I realize you can just buy them (which we have and thus, have a stock that we can propagate), but considering how expensive it is to buy them, and how cheap it is to grow bacteria, it seems silly to continue to buy them. So have people done this? Are there any special considerations for these cells versus other cells?

They behave like any other cells. So, if you can make good competent cells of other strains, you can make good cells from this strain. Usually, people have trouble with this, and wonder why their "ligations" don't work, when it is really that their competent cells are lousy.
http://openwetware.org/wiki/TOP10_chemically_competent_cells

That's what I figured. Do I add chloramphenicol to the LB to select for these cells or no?

The cells are not resistant, so, no you should not add chloramphenicol when preparing the cells.

Hi kfunk106!

I am thinking of preparing ccdB survival competent cells for our lab use. I read your discussion in the forum. Wonder, how did it go? How much was the efficiency you could achieve?
Thank you in advance.
cheers

I think it went ok. Probably better to just buy them and not deal with it though.

Following you guys' discussion,

I ran out invitrogen DB3.1 stock, and want to make my own ccdb competent cells. Tried once, but efficiency for ccdb vector is extremely low despite high competency for pUC19.

Wonder whether I should add streptomycin to maintain genotype/ccdb resistance of DB3.1 due to its StrpR genotype.

Are there any other tricks to maintain ccdb resistance?

Thanks so much for advice,