Glycerol Stock - (May/01/2014 )

I'm working on my bachelor thesis and finally got some good clones to continue with. I wanted to store them in glycerol stocks, but I guess  I missunderstood my adviser. I took 1400µl glycerol and 600µl overnight culture (so it's 70:30). But from others I know that they take more culture than glycerol... so I wonder if this much glycerol is very bad for my cultures? It's 80% glycerol.

Please excuse my english!

80% glycerol has a very high osmotic potential - it will probably dehydrate your cells and damage them irretrievably.  You should have 15-30% glycerol in your stock.

bob1 on Thu May 1 23:57:56 2014 said:

80% glycerol has a very high osmotic potential - it will probably dehydrate your cells and damage them irretrievably.  You should have 15-30% glycerol in your stock.

Hi bob1, what about 50% glycerol in LB media? Is that considered ok?

You'll probably find that 50% glycerol is too strong.

Here's what Sambrook et al,: Molecular cloning: a laboratory manual, has to say (abridged):

1.5 ml culture, add 0.5 ml 60% sterile glycerol

Vortex

Snap freeze

Transfer to -80 freezer.

Just to be clear, bob1 is talking about the final concentration of glycerol. 

Our lab uses a stock of 50% glycerol in LB, but that's the stock. We add equal amounts of that to our culture for a final concentration of 25% glycerol.