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title: "TheNessLab : Freezing/Thawing protocol"

1. TheNessLab
2. The Ness Lab Wiki
3. Chlamydomonas information and protocols

TheNessLab : Freezing/Thawing protocol

Created by Josianne Lachapelle, last modified by Ahmed Raza Hasan on Oct 13, 2019

Prior to freezing

Take a few colonies from agar plate and inoculate into ~2ml Bold's liquid (or more, depending on how many replicates you are freezing). Grow for a few days (usually 3-4) until green growth is visible.

 

Setting up

• Leave cultures on lab bench (in dim/ambient light) for at least a couple of hours before starting the freezing protocol.
• Make cryopreservation liquid - 10% volume: volume with Bold's liquid (for example: add 5ml of Methanol to 45ml of Bold's in a falcon tube).
• Filter sterilise the cryopreservation liquid using 0.2μm filters
• Label tubes and add 0.9ml cryopreservation liquid to tubes.
• Need boxes to hold tubes (cryo boxes of 81 tube capacity).

 

Freezing process (work with lights off in the flow hood)

• Add 0.9ml of the grown liquid culture to the cryotubes containing 0.9ml of cryopreservation liquid in low light levels.
• Vortex, and place cryotube into 'Mr Frosty' freezing container and put container into -80°C freezer for 4 hours.
• Take tubes out of 'Mr Frosty' and plunge into liquid nitrogen for storage.
• Note: After every 4 or 5 use of a Mr Frosty, the IPA liquid in the Mr Frosty should be changed

 

Thawing and reconditioning frozen stocks

• Take tubes out of liquid nitrogen.
• Place tubes in water bath at 35°C for 5 mins. Be wary of lids potentially popping off into your face. To avoid this I put the cryotube box with lid on into the water bath with tubes inside.
• Pipette entire contents of tube (1.8ml) into 10ml of Bold's liquid in a sterile universal and incubate for 4-5 days (or 200uL onto agar, or 300uL into 1.7 mL Bold’s in 24-well plate).

Strains available in the lab

Ness Lab Nitrogen Dewar

Document generated by Confluence on May 22, 2024 11:44

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