"How to add strain and grow from strain collection"

Step-by-step guide

What should go in the strain collection?

  • If you have strains that meet any of these criteria, put them in this collection:
    • sequenced genomes
    • published
    • sent by collaborators
    •  you have sent the strain to a collaborator


How to prepare samples to be stored?

  1. Verify the identity of your strains by 16S/ check plasmids by sequencing. Store isolates from single colonies, and perform isolation streaks to ensure that no mixed isolates are stored.
    1. we maintain a backup of the strain collection. Please store your strains in both collections. One tube should go in the -80 in 428, one tube should go in the green -80 in 433.
  2. Store in 17-25% glycerol in a cryo-vial.


Which tubes?

Use Wheaton freezer vials. Different colored caps correspond to different types of strains:

White caps=Natural isolates

Blue caps= E. coli strains used for molecular work

Red caps= Genetically modified strains.


Protocol:

  1. Grow overnights in rich medium + selection if needed
  2. Record your strains in the strain collection excel sheet. The excel document is in dropbox>cordero lab>data>STRAIN collection>OXC_STRAIN_LOG. We currently maintain two tabs- one for natural isolates, and one for E. coli/GMO strains. This is because the metadata associated with either type of isolate differs.
    1. Check for the highest # strain on both tabs of the excel sheet
    2. Please enter as much metadata as possible into the fields.
    3. The location of linked data files (like phenotypic information) should be noted.
    4. Close the strain log when you are done. If two copies are simultaneously being edited, a conflicted copy will be created.
  3. In the morning, add 1 mL 50% glycerol to a cryo vial. Add 1 mL of your overnight, and vortex to mix.
  4.  Label 2 tubes for each strain (main collection + backup) with:
    1. (top) # in strain collection
    2. (side) strain name/date
  5. Place main/backup vials in the appropriate -80s. Our strains do not need to be flash frozen before storage.


How to handle strains when needed for your experiments?

  • You can tap into the original strain collection EXACTLY once, and this is to make your own working stock. From then on, you need to work with your own stock.
  • When you work with the vial from the original collection you need to make sure the vial is kept as cold as possible (warming will negatively impact strain viability).
  • How do you keep the vial cold?
    • If you are working with one or two vials, place the vial /vials on a pre-chilled cooling block and minimize the time the block and vials are out to the absolute minimum (ie work fast).
    • If you need to work with more vials or anticipate the work will take longer you need to use dry ice. It is not good practice to take vials out one at the time from the -80C since open and closing the -80C repeatedly will cause a drop of temperature that will affect all samples in freezer.
  • If you are unsure on the best way to handle the strain collection talk to the lab manager or a lab member who has considerable experience handling frozen stocks.



Keep vials cold!

Tap into main collection only ONCE!

Store your strains in both back up and main collections.