Starting cells for making one's own competent cells
Posted 12 January 2011 - 08:14 AM
Posted 13 January 2011 - 02:50 AM
Posted 13 January 2011 - 10:18 AM
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)?
Edited by seanspotatobusiness, 13 January 2011 - 10:25 AM.
Posted 13 January 2011 - 02:18 PM
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.
Posted 14 January 2011 - 02:50 AM
None of the following protocols state which cell types they are best applied to, nor the expected efficiencies:
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 [F´ proAB lacI q Z∆M15 Tn10 (Tet r ) Amy Cam r ]. ‡ (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?
Edited by seanspotatobusiness, 14 January 2011 - 02:51 AM.
Posted 15 January 2011 - 04:34 PM
Posted 15 January 2011 - 06:33 PM
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...)
Posted 22 January 2011 - 08:59 PM
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.