6 replies to this topic

[cathy]

dear all,
I have colonies in plate after cloning. then i picked up colonies for growing small culture to do miniprep and the insert is there. then how should i store it? i have remained 0.5ml culture. should i using it to make a glycerol stock? what percent glycerol i should use? or using this small culture to do a midi prep then store the plasmid?

Million thanx

cathy

[fred_33]

hi
this page of bioforum will help :

Storing of miniprep or maxiprep plasmids

fred

[george@CASE]

i usually make glycerol stocks of important cultures (cultures containing good intermediate vectors or final constructs).

I then do restriction digest on my minipreps (from the same culture), and then toss out stuff that don't have the insert/wrong orientation.

[cathy]

thanks fella
really helpful!!!
by the way, how do you make the glycerol stock? 85%glycerol?

[fred_33]

hi
i've read that you should have at leas 30% glycerol. But for mine, i pick 1ml of culture and complete the so called "eppendorf" (to 1.5 - 1.6ml) with anhydrous glycerol.

[george@CASE]

hmm, funny... I just looked at my protocol and I used 850uL culture plus 150 uL glycerol (less than 30% glycerol). Now, this protocol is from someone else, so don't take it as the correct one. Anyway, I've restreaked from my glycerol stocks and they come out fine.

Hmm, I better take a closer look into this.

Edited by george@CASE, 30 March 2005 - 01:15 PM.

[methylnick]

george@CASE, on Mar 30 2005, 02:15 PM, said:

hmm, funny... I just looked at my protocol and I used 850uL culture plus 150 uL glycerol (less than 30% glycerol). Now, this protocol is from someone else, so don't take it as the correct one. Anyway, I've restreaked from my glycerol stocks and they come out fine.

Hmm, I better take a closer look into this.

<{POST_SNAPBACK}>

george, that is what my protocol says also, 15% glycerol, and it works fine for plasmid clones and BAC clones.

Nick