Frozen transformed bacterial cells problems

[chancer]

1. Should I sterilize the cap before opening the cryo vial? Should i also sterilize the opening before recapping it? And what should i sterilize it with?

2. How can i make a new culture from the frozen transformed stock? Should I use a pipette and take some of the frozen transformed cells directly into a new liquid medium and shake it overnight? Or should i add the frozen transformed cells directly on an agar medium plate? Should the cells be thawed first? Can i freeze the rest of the cells back in the freezer or do i have to use them all at the same time?


Thanks for any inputs.

[canalon]

1. Should I sterilize the cap before opening the cryo vial? Should i also sterilize the opening before recapping it? And what should i sterilize it with?

If you have external screw cap tube, no need to sterilize. Just limit the opening time...

2. How can i make a new culture from the frozen transformed stock? Should I use a pipette and take some of the frozen transformed cells directly into a new liquid medium and shake it overnight? Or should i add the frozen transformed cells directly on an agar medium plate? Should the cells be thawed first? Can i freeze the rest of the cells back in the freezer or do i have to use them all at the same time?
.

To start a new culture the best is to scrap some of the frozen culture and plate it on selective agar. And restart any liquid culture from an isolated colony. Use a bacterial loop for this is the most convenient.
If you want to keep your frozen sample avoid as much as possible any thawing (take it out of the fridge as shortly as possible, always keep your tubes on ice...). Ideally you should have 2 frozen stock: A working one(or more), that you can throw away in case of thawing, and a stock/backup that you use only to prepare working batch once in a while.

HTH

Patrick