Smolke:Protocols/Freezer stocks

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
• 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
• 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