Mini prep streaking - basic question (Oct/11/2010 )

I haven't done this in a while:

I have colonies to pick to do a mini-prep which I will eventually do a maxi prep.

Basically I did a gateway clonase reaction and transformed competent cells and now I am selecting 3-5 colonies to test via restriction enzymes after doing a miniprep. I know I have to somehow re-streak each clone so that when I choose which ones are correct, I can go back and find them again for a maxi prep.

Is that done when I pick a colony and add it to 2 ml LB+amp, leave it overnight and then streak a plate? Or do I just dot a plate? Or is it done before th overnight growth?

Thank!

Here what I usually did.

Before picking a colony, mark with your sharpie so that you know which one is the one you already picked and later you can go back and pick that again if that is a right one.

After picking 1 colony and grow them in 3-4ml LB with Amp overnight. Do a digestion to see if you got the insert. If you do, you can just use the left over culture and transfer 5-10ul into new LB culture and incubate for maxiprep. Or you can just go back to your plate and pick that colony again for the maxiprep.

And dont just pick the whole colony, use your pipet tip and just barely touch the colony is more than enough bacteria

You could also make glycerol stocks of the clones, and throw out any that are incorrect later.

For 3 to 5 well-isolated colonies, I would loop them off the transformation plate and streak each to its own plate. Grow this plate overnight and use some growth for an overnight 5 ml broth culture for a mini-prep. The next morning, I'd do the mini-prep, digestion, and run the gel. Then I'd start fresh 100 ml overnight cultures of any you'd like to keep in the afternoon. The next morning, I'd take a bit of the 100 ml culture and make glycerol stocks, and use the rest of the culture for a maxi-prep.