UA Biophysics:Protocols:Stocks

FIRST DAY

1. Bacteria taken from either -80 tubes, or remaining stock and streaked onto LB agar plates (with antibiotics if resistance is in the strain)
2. Grow plates overnight in hot room, collect early next day to get small colonies

SECOND DAY

1. Select small single colony and add to 10ml LB (with antibiotics if resistance is in the strain) portioned into conical flask or glass bottle
2. Place flask in hot room on shaker for overnight growth
3. Collect flask early next day

THIRD DAY

For Long-term Storage

1. LB:Glycerol(50%) 1ml:1ml ratio
2. Portion grown bacteria into cryovials (2 ml each)
3. Freeze bacteria in liquid nitrogen
4. Store in -80C freezer.

For fast recovery stock:

1. LB:Glycerol(50%) 3ml:2ml ratio,
2. Portion grown bacteria into eppendorfs (130ul each)
3. Freeze bacteria in liquid nitrogen
4. Store in -80C freezer

RECOVER

Recover bacteria from glycerol stock: to open the tube and use a sterile loop, toothpick or pipette tip to scrape some of the frozen bacteria off of the top. Do not let the glycerol stock unthaw. Streak the bacteria onto LB agar plate.

Recover bacteria from fast recovery stock: transfer the complete volume to a tube with LB (10 ml) using a pipette.

Grow bacteria overnight at the appropriate temperature.

NOTES

The stock is stable for years, as long as it is kept at -80. Subsequent freeze and thaw cycles reduce shelf life. Stock has to be placed in liquid nitrogen, when it is out of the freezer.

LB:Glycerol(50% v/v) have to be autoclaved.

AMP AND KAN CONCENTRATION

1:1000 dilution of 100mg/ml amp 50mg/ml kan

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