5 replies to this topic

[criscastells]

Hi everybody,

I am new at clonning but I have obtain (not without a lot of problems ;P) transformed cells with my insert of interest. Right now I have this clones in an agar plate and now I want to prepare cultures with grlycerol and freeze them. If this LB plates are a little old is it possible that the cells have lose the vector?

The other question is if it is better to prepare a new plate with isolated colonies and freeze a unique clon or I can pick a group of colonies of my master plate and grow in liquid LB to freeze.

Thanks you very much in advance for your help

[jerryshelly1]

How old is a little old and how was it stored.  You could run into the risk of contamination, but hopefully your drug resistance will cure that.  Just try a quick isolation, miniprep the sample and reconfirm with digestion.  If it is still there, you can use your previous inoculated media and re-grow it.  good luck

[criscastells]

Thanks for your useful and really quick answer!

[Missle]

Sorry for an obvious question but...your LB plates have antibiotic, right?  If not, your vector is gone.  I also wouldn't mix multiple colonies from a transformation.  You're better off to pick a single colony, grow w/antibiotics, and freeze that down with glycerol.  Better be safe then sorry when it comes to cloning!

[criscastells]

Yes, my plates have antibiotic. I know my vector is there by colony PCR. I pick a single colony to prepare a master plate from the colony PCR and now I want to save the positive clones and I don't know if it is better to prepare a new plate and pick a single colony or I can prepare the stock with the master plate. I think I will pick a single colony.

[bob1]

The ideal technique is to re-pick the clones and streak out on a fresh plate, then pick a single colony from that plate and use that to grow up and make a glycerol stock.