Making Competent Cell

[kenpier]

Hi,

Anyone here have a good protocol in making chemical treated competent cells fast?

Currently using rubidium chloride method.

[canalon]

RbCl is exactly what I use when I want to obtain chemically competent cells quickly with a good efficiency.
Following a protocol similar to this one: http://130.15.90.245/e__coli_competent_cells.htm

What is wrong with it?

[kenpier]

The competency level of the cell don't always hit 1x10exp8 to 1x10exp9 cfu's/ug.

Sometimes is just too low, result are not always consistent. Is there anything to take note?
I know that TFb1 and TFb2 need to dissolve individually before mixing.

[canalon]

I must say that I never measured the competency level, and in most case got my transformants (as for most of the other cases, that is common when you try to express a protein that is toxic for your bacteria).
I have been known to "steal" from TA cloning kits the competent cells that came in the package (we had a lot of that because generally they were not what we wanted to transform our constructs in) as they are a bit more consistently successful.

But for TFb1 and 2, they were prepared in a batch in advance and stored in the fridge fully dissolved and ready to use (they are really stable).

[kenpier]

When preparation of competent cell, which freezing well be better?
Dry Ice or Liquid N2?

[JackBean]

the cells need to be frozen as fast as possible, so that the water crystals will be small. For that reason is used nitrogen usually.