E. coli Strains - (Feb/13/2009 )

This is a follow up to my dead E. coli problem (posted already on the forum).

I have been using a kit from Clontech called in-fusion 2.0 for cloning, produced my clones and transformed them into their "super competent" FusionBlue E. coli cells. This worked fine and produced many colonies, but I am keen to get back to using a more common generic cloning strain used in our lab already, or common in other labs.

I have tried to transform the same clones into DH5 alpha cells, but no colonies grew. For these reactions you need "super competent" cells (according to the website), with a transformation efficiency of greater than 10^8 cfu/ug DNA (that doesn't mean anything to me, but if it does to anyone else?....).

I was wondering if anyone could suggest any strains I could try, I've been told to try Invitrogen's Top10 E. coli or Excel-1-Blue/NovaBlue (Transformation efficiency: 10^9 cfu/μg DNA).

Are there any others which could be useful?

If you do decide to go with the TOP10 cells from Invitrogen, the chemically competent cell preps they sell work great. I've tried to make my own electrocompetent version of these cells though and I don't get nearly as many colonies (they seem to be equivalent with DH5alpha).

Hey

I use XL1 strain and it works very well. Infact for SDM they work better than the comp cells provided for the kit.

TC

How are you making your competent cells? I have used DH5alpa made competent by the RbCl2 method successfully with the in-fusion method...

We use a commercial strain from invitrogen, supposed to have "subcloning efficiency". I have just looked up the strain and the competancy is 10^6 cfu/ug DNA, so possibly not enough for the in-fusion reaction. I will try a few of our top10 and XL1 strains in house and see if I can buy in a strain of DH5 with a higher competancy!