need to make a new glycerol stock - (Apr/24/2015 )

Hi,

Yesterday I streaked out E.coli containing my plasmid onto LB+Amp100 plates from the glycerol stock, and this morning the colonies had irregular shape and they looked bigger than O/N colonies should (incubated for ~15 hrs at 37'C). The glycerol stock is not mine and I know that previously it got thawed a few times. However, I have a plate from about 24 days ago that I've kept the plate at 4'C wrapped with parafilm and it has nice single colonies. So I was wondering what is the best way to make a new glycerol stock? Should I just take a bunch of colonies from the old plate and re-streak them onto a new plate and then make a liquid culture, or can I directly take one single colony from the old plate and grow the culture for the glycerol stock?

The plate can still be ok, but you need to restreak them on a new plate.

The glycerolstock: hard to tell if there is something wrong... Bigger? Its very subjective...

But do not go directely from the old plate to a liquid culture.

Hi pito

I'm just wondering if there'll be an issue in case of having a direct colony (from old plate) to a liquid medium.

Thank you

The general idea behind Always going to plates first is simply to check for contamination and get a single colony (fresh one).

Of course, the old plates also contain single colonies and you can see if there is contamination or not, but they are "old" (as it is an old plate).

It is an extra step, but one that can save you lots of time if it turns out later (when used the old plate) that the old colony you used was bad....

Of course: you can discuss about what old is and how many days/weeks .... , but Always best to work with fresh material (few days old) just to save you the troubles afterwards.

Thank you!