why OD600=0.3~0.4 is important? - (Apr/05/2008 )
When prepare competent E. Coli using Calcium Chloride, why the OD600=0.3~0.4 of E. Coli is important?
Thank U very much indeed?
You want as many cells as possible, but you don't want the cells to transition into stationary phase. That happens somewhere between 0.3 and 0.7 OD, so people tend to be conservative about amount of growth allowed. The OD measurements are not very consistent between specs, so your OD 0.3 may be quite different from someone else's.
Thank You very much indeed, Phage434
I prepared a vial of E.Coli(TOP10) on Apr. 4th，When I pick a single clony into a 50ml centrifuge tube containing 5 ml LB, and incubated it at 37oC on a shaking incubator at 225rpm, 3 hours later(OD=3.0 according to Molecular Cloning), I get no E.Coli after centrifugation. I was blue then. I used another overnight LB which of course had E Coli in them(but OD might >0.3). I used this Ecoli to prepare competent Ecoli, I get amp-resistant E.Coli
what if I use Ecoli which is in stationary phase?
I appreciate your advice.
If you got transformation of your cells, carry on happily. The goal of the 0.3 OD measurement is to improve the transformation efficiency. This is typically not important for transforming existing plasmids into cells, but can be very important in cloning reactions where you are trying to transform cells with ligation products. The CaCl2 cloning tecvhnique is low efficiency, and probably not suitable for most ligation cloning. Far better approaches exist, such as the CCMB80 chemical transformation technique, or electroporation.
thank you phage434
i'm going to do a PCR clone , and transform this ligation product into E.Coli. I do not get any transformation clone, you mean I should get rid of my competent EColi and buy some competent cell?
We don't buy competent cells any more (we would be broke). We make them, see here:
Can I substitute SOB with LB in the protocol you mentioned early
I don't know. Probably, but these protocols are quite fussy. If you have 2XYT that would probably be a better substitute.
many thx phage434
i put a amp-resistant plasmid into TOP10
but it grown on LB plate containing 50ug/ml Amp 24 hours later
I observed it at 12hours, nothing is visible
is it my desired TOP10 containing the right plasmid
about 15 colonies per plate
I picked one colony last night and incubated it at 37C shaking incubator 225rpm
I got turbid LB this morning
I extracted plasmid using OMEGA plasmid kit
how do i further identify this pasmid?
thank you in advance