Checking for efficieny competent cells - (Mar/28/2014 )
I saw this post in another topic (http://www.protocol-online.org/forums/topic/31960-failed-cloning-different-readings/#entry166263):
phage434 on Tue Mar 25 18:00:51 2014 said:
I meant (2). Establishing the competence of your cells and transformation techniques is very important, because ligation, even under good circumstances, produces low numbers of correct circular molecules. Competence is usually expressed in colony forming units per microgram of DNA. If you dilute your uncut vector to concentration of 10 pg/ul (10^-5 ug per microliter) and transform with 1 ul, you can determine your efficiency. Good cells and technique will give 10^8 to 10^10 cfu/ug, or 10^3 cfu to 10^5 cfu in the transformation I suggested. If you are getting less than 100 in that test, you will have trouble with ligation reactions no matter what else you do. People often assume that because they can transform prepared miniprep plasmid that their cells are sufficiently competent. This is definitely NOT true, and is a leading cause of problems.
Now I wonder: how many people here really check for this efficiency in this way?
I only do the basics of cloning to make various expression constructs, so I never check the efficiency of my cells. I just make sure to make them very carefully, and not cut corners while doing so. If I don't get colonies after a ligation, then it's usually not my cells that are the problem but rather poor cloning prior to transformation or dead ligase. I find this is usually the case with many members of the lab as well, except they want to blame bad competent cells for their lack of colonies when in reality it was shotty work in the upstream cloning steps.
If in rare cases I'm ever hesitant that my chemically competent cells don't have enough "juice" for a transformation (like for double transformations), then I just electroporate. Electrocompetent cells are easy enough to make (3-4 washes in 10% glycerol). I just keep a stash in the -80C for the rare occasion that I need them.