Glycerol Stocking

After successfully sequencing a bacterial plasmid, we stock it for long-term storage. Bacterial plates keep well for ~1-2 months at 4ºC, but may dry out or be contaminated if stored for longer.

Note

This protocol describes how to stock plasmids in the lab’s shared pKG database. For personal glycerol stocks (e.g., plasmids waiting to be tested in TC before adding to the database), one vial per plasmid is sufficient. These can be stored in personal boxes in the -80ºC.

  1. Add the plasmid to the database in Quartzy. See instructions on this page.

Important

Add plasmids to the database before stocking! That way, you don’t run into issues with overlapping numbering if someone else is also stocking around the same time.

  1. Culture 4 mL of the bacteria clone with the plasmid in LB Broth with the correct antibiotic. Shake at 30ºC overnight.

  2. The next day, add 2 mL of 60% glycerol to the 4 mL of culture. Mix well.

  3. Aliquot 1.5 mL per vial of the resulting mixture to three cryovials.

Note

Avoid overfilling the cryovials, as this makes them hard to open. The maximum capacity is 1.8 mL to allow room for expansion upon freezing.

  1. Label each cryovial with pKG#, date, initials, and description of the construct. Labels can be printed using the LabBot app on slack “Print Labels”. (If it does not show up begin to type “/labels” in the Slack search bar to find it.) You must be connected to MIT Wifi for this app to work.

  2. Store cyrovials in the designated boxes at -80ºC. Put one cryovial per box.