growing E coli from a starter culture

[lajoien]

My professor has previously made a starter culture that is in the freezer of e coli cells. How exactly do i go about growing a culture can i just use LB broth in a flask or do i have to plate some of the cells LB-AMP before growing them in LB broth overnight.

[lajoien]

my email address is lajoien@stu.easternct.edu if anyone knows a protocol to follow that would be great

[blcr11]

I guess the "proper" way is to gouge out a bit of the culture and streak it onto plates (with the appropriate selective media), grow the plate overnight, and then start a fresh culture from a single colony on the plate. It also depends on whether you’re trying to make fresh plasmid, or grow cells up for protein expression. If you’re after plasmid, I would recommend streaking first. If the frozen culture is a plasmid in an expressor line (like BL21 or its derivatives), and you’re lazy like I am, you can make a fresh starter culture directly from the frozen stock without troubling yourself with re-plating first. So it depends on how much of a purist you are, I guess.

[canalon]

I would agree with blcr11:
-If you are in a hurry, you can go directly from glycerol to broth.
-but if you have enough time streak on a plate and restart from an isolated colony.

and ampicillin stinks as a selective antibiotic. Although I suspect you did not have much choice. more stable antibiotics that are not degraded are a better choice. But for that same reason I would definitely try to go with a first isolation/selection step before restarting a culture.

[blcr11]

Yeah. Use carbenicillin in place of ampicillin whenever you can. It’s more expensive, but much more stable.

[lajoien]

actually i am trying to grow up a culture to purify the protein alkaline phosphatase. at least that is what my protocol calls for. so lets say i was to just make a starter culture in a the LB broth ; how exactly do i go about doing it like is there a method to follow or like a standard website.

[canalon]

GOOD technique:
From the starter culture isolate colonies on LB+Amp (or carbenicillin) plate, pick one nice isolated colony and put it in fresh LB broth containing Ampicillin (or carbenicillin), and have fun extracting your protein.

QUICK technique:
Directly from the starter to the broth, hope that the selection was good and that it will be good enough to be maintained in the broth (a sacrifice to the microbiology gods is usually recommended, as a post-doc I tend to use undergrads, but you can choose something else ;-)) and extract your protein as above.

It really depends on the amount of time you have, your experience with the stability, or the lack of it, of your plasmid, your trust in the starter, and your luck.

[blcr11]

You want a blow-by-blow description of how to do those things, then, I gather. You can browse Protocols Online if you want:

http://www.protocol-online.org/prot/Molecular_Biology/

You can try and get ahold of a copy of Maniatis’ manual on Molecular Cloning, though it may be seriously out of date by now. Those would be two fairly comprehensive sources.

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