Re: Back to Transformation! (Was Re: [DIYbio] Re: Which microscope?)

You're going to need LB liquid too for the overnight and 3-5 hour culture prior to transformation, as well as 2ml for the post-transformation recovery period... so make about 150ml total LB, split into three 100ml flasks each with 50ml in (autoclave fluid at half capacity of container). Add the agar to one of the three flasks before autoclaving, then after autoclaving when its cool enough that the flask is comfortable to hold with your bare hand, add the ampicillin to the agar flask only, and mix to swirl, then pour into the petri dishes (25ml per dish should work).

50ml LB with a scrape of e.coli culture and incubate in shakers incubator overnight, or about 12-18 hours.... Then add about 5 ml of that in 30ml of fresh LB and incubate in shaker for 3-5 hours just prior to transformation. You'll need a centrifuge if you want to do CaCl2 method, basically just take 1ml of the 3-5 hour culture, and centrifuge it in a microtube, the cells collect at the bottom, and dump off the LB media, replace with 1ml CaCl2, vortex to resuspend, centrifuge again, dump off liquid, resuspend in fresh CaCl2, repeat maybe one more time and then let chill on ice... Add DNA, chill, heat shock, add 2ml fresh LB, incubate in shaker for 1 hour, plate on LB agar with ampicillin... (This is the basic idea, protocols may include additional chilling steps, etc). Read the manual I posted from my school.

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