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 I believe the back-dilution is to ensure that you freeze the cells when they are in the exponential growth phase and are the healthiest. If the stocks made from overnight culture grows just fine I suppose there's no problem with that. --YC The back-dilutions are to ensure that you make freezer stocks of cells when they are in late exponential phase and healthiest. This way you have more of your cells healthy and alive when you make the freezer stocks (with the idea that they will last longer and grow up better from the freezer stocks). Typically, I always made freezer stocks in the late exponential phase (ODs for this are different for yeast and bacteria). --CDS 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 NB - this same protocol works for yeast and DNA freezer stocks. -Josh