Starting cells for making one's own competent cells - (Jan/12/2011 )

When making chemically or electrically-competent cells, is it usual to start with non-competent cells? I've been unable to locate such cells. Alternatively, is it usual to buy competent cells, expand them and make them competent again, according to your needs?

Yes, usually you would buy competent cells with the genotype of your liking, expand them and render them competent again. Be sure to keep them at 4C all the time!


rsm

Thanks, RSM

I just thought of something else - could you use cheap chemically-competent DH5-a and make them into decent electro-competent cells? Do you suppose there is any difference between cells expanded from subcloning efficiency DH5-a and those expanded from super-efficient cells (or are they all the same, only differing in their treatments)?

You don't need competency in the cells to prepare them to be competent after all, the first person to prepare competent cells must have used "non-competent" cells in the first place. It does help to have a strain (such as DH5aplha) that is easy to make competent though.

Be aware that there are specific methods for preparing competent cells that depend on the method of transformation to be used and on whether you want to store the cells.

Thanks, Bob. What about all the different protocols for making electro-competent cells? I suppose some will work better with certains cells than others?

None of the following protocols state which cell types they are best applied to, nor the expected efficiencies:

http://openwetware.org/wiki/Electrocompetent_cells
http://openwetware.org/wiki/Knight:Preparing_electrocompetent_cells
http://openwetware.org/wiki/Belcher/Knight:_Electrocompetent_Cells
http://openwetware.org/wiki/Richard_Lab:Preparing_electrocompetent_cells


Would it be worth starting with Xl10 Gold cells?

From the Stratagene manual for these cells:

"Genotype of XL10-Gold ultracompetent cells: Tet r ∆(mcrA)183 ∆(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte . ‡ (Genes listed signify mutant alleles. Genes on the F´ episome, however, are wild-type unless indicated otherwise.)

"XL10-Gold cells are tetracycline and chloramphenicol resistant. ‡ XL10-Gold cells are deficient in all known restriction systems <∆(mcrA)183 ∆(mcrCB-hsdSMR-mrr)173>. The strain is endonuclease deficient (endA), greatly improving the quality of miniprep DNA, and recombination deficient (recA), helping to ensure insert stability. The Hte phenotype increases the transformation efficiency of ligated and large supercoiled DNA. The lacIqZ∆M15 gene on the F´ episome allows blue-white screening for recombinant plasmids".

I don't know whether those mutations are common or whether they'll even help without the proprietary treatment that Stratagene can give them?

Thinking about it, if those XL10 Gold cells are proprietary, their licence for use probably prohibits trying to expand them and make them competent yourself, anyway. Unless I recieve information telling me otherwise, I'll probably just start with DH5-a because they're cheapest and try others if I can't acheive satisfactory efficiency.

Any of the protocols should work for preparations, some produce slightly more efficient cells than others... but it varies a lot depending on who makes them.

You should expect to get 10^8-10^9 colonies/ug/ul (or whatever the units are) for heat shock competent cells, I think electro-competent cells should give you more.

You wouldn;t be the first to expand propietary cells, just be aware that some strains may not do well as they are prepared by mating or something to stop you trying to expand them (I don't really know, I'm not a microbiologist...)

To put it simply.. it is the treatment method that makes the cells competent, super competent or ultra competent. This is what you are paying for... the preparation method and convenience of not having to spend the time yourself.

So you can use any strain with the phenotype that you want to make competent cells. The phenotype influence what kind of plasmid (size, repeat structure) you can maintain in the cell or how well it can express a protein.

It is the preparation method that makes the difference between competent and ultra competent cells. If you want to save money you can make your own competent cells, and a normal prep of electrocompetent cells is usually 10^8 colonies per ug DNA. (Unless you can find somebody who actually took the time to sit down and rediscover the right conditions to make ultra competent cells).

All strain can be grown and used to make home made competent cells. If said strain could not grow, it would be pointless as you would never be able to use for plasmid production or gene expression. You need more than a single transformed cell.