A glycerol stock of bacteria is both great for long-term storage and very convenient to recover.

1. Freezing down bacteria for cryopreservation

1. Pipette 0.5 mL overnight culture of bacteria containing the desired plasmid into a sterile 1.5 or 2 mL tube.

   • We usually use the 2 mL cryogenic vial, but in practice any sterile tube works since the vial is for -80°C storage rather than liquid N₂ storage.

2. Add 0.5 mL 50% glycerol (by volume) to the vial. Mix by inverting for a few times.

   • To make 50% glycerol, pour sticky 100% glycerol into a 50 mL tube to ~20 mL mark. Add equal volume of water by pipetting. Rotate the tube until the solution is homogenous. Filter sterilize.

3. Store in -80°C.

2. Streaking bacteria from frozen stock

1. Warm up a selection plate to room temperature.

2. Take out the frozen stock from -80°C onto a box of dry ice to prevent it from thawing.

3. Poke a sterile 1 mL pipette tip into the glycerol stock and streak attached bacteria onto the selection plate by drawing zigzag on the plate for a few turns. Restrict the drawing within half of the plate.

   • To avoid scratching the plate, place the tip at an angle to the plate and draw lightly.

4. Use a new sterile 1 mL pipette tip to draw zigzag out from previously drawn lines into untouched area of the plate.

   • The first round of drawing usually have too many bacteria so it will unlikely to grow single colonies. This second round of drawing aims to spread a tiny amount of bacteria by touching previous lines to get single colonies.

5. Incubate the plate at 37°C overnight or 30°C for 24 hours (for plasmids with high risk of recombination such as lentiviral vectors).