Smolke:Protocols/Freezer stocks

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How Grow up overnight liquid culture Back-dilute 20 ul into 5 ml in the morning Grow until OD 0.7-0.9 (4-6 hrs depending on cell density and viability during back dilution) Add 1.5 ml culture to 0.5 ml 60% glycerol in cryovial Final concentration should be 15-20% glycerol. I use 1 mL culture in 0.5 mL 60% glycerol and the freezer stocks are fine. -Josh Mix by inverting tube Store at -80C For every plasmid registered in lab data base, there should be 1 working stock (in the working -80C) and 2 backup stocks (in the backup -80) When I generally make 1 freezer stock by back-diluting from the overnight culture that I use to prep plasmids for sequencing. This way I am certain that the freezer stock is identical to the sequenced sample. Once the sequence has been verified, I would then go back and make the backup freezer stocks by inoculating from the first stock I made. This step also serves to verify that the first stock would grow properly in liquid culture. --YC I've never understood the reason for back-diluting before making a freezer stock. You're just adding more time for the plasmid to mutate before being frozen. I make my freezer stocks directly out of my overnight cultures and they work fine. -Josh If I had reasons to doubt the sequence accuracy and did not wish to make a lot of freezer stocks before verifying the sequence, I would inoculate from my restreaked plate right after the sequence has been verified (the plate is generally only 2 days old at this point). If I cannot make the freezer stocks before the plate is ~1 week old, I would transform new cells with sequenced plasmids and inoculate from the newly transformed cells. --YC Notes I also keep a backup of every plasmid I have constructed (in DNA form), and this is generally the same sample that I had submitted for sequencing. This may be more cautious than is necessary, but I don't think it hurts to be careful. --YC