Storing Organisms in the -80C freezer (iGEM)

Updated 6/10/2019 09:14 PM

by SoundBio iGEM (created by Yoshi Goto)

Introduction

In order to store strains for nearly decades, you can preserve them in glycerol and store them in the -80C freezer at SoundBio if you have permission. This protocol describes exactly how to do that. This is a shared space and strains can serve as the foundation for someone's research. Thus, in order to store them, one must follow a specific systematic process to avoid destroying the strains of other lab members and their own strains.

Materials

Equipment

2mL sterile cryotubes
3/8 inch diameter "MICROTUBE TOUGH-SPOTS" white cryotube labeling tape
White labelling tape from on top of the microwave
"SARSTEDT permanent" marker for labelling cryotubes
P₁₀₀₀ pipette with tips
-80C freezer "Yeti"
Laminar flow hood
Gloves

Samples + Reagents

At least 2mls each desired strain from an overnight culture
80% sterile glycerol aliquot from the storage room (make sure your aliquot is labelled with your name and you are only pulling from your own aliquot)

Procedure

Storing E.coli Strains in the -80C freezer

Locate the "iGEM (GM) Organism Database" Google spreadsheet.

Fill in the desired information about your strain, recording the SB number if your strain was pulled from the "-80C SoundBio Official Organism Database". Your personal strain number should start from 1 and increment by 1.

Label each 3/8 inch diameter "MICROTUBE TOUGH-SPOTS" white cryotube labeling tape with your respective strain number. i.e. if your strain number is "GM04", label your cryotape with "04". Use the "SARSTEDT permanent" marker for labeling the tape.

Label the inside of the tube cap itself with your number. This is to ensure that if the tape comes off, you can still find a number label. Let the ink dry for 1 minute.

Place labelled tape all the way into the lid of each respective sterile 2mL cryotube, making sure the strain number is completely visible from the top.

Use the end of the marker to push in the labeling tape. The marker just fits inside of the tube cap.

Label the white label section found on the side of each cryotube with your full strain number and date ** using the "SARSTEDT permanent" marker.

** i.e. "GM01 10/31/18"

To determine the volume of 80% glycerol stock solution to add to your strains for cryopreservation, conduct the following calculation:

(Volume of 80% glycerol to add) = (desired glycerol percentage)* (2mL)/(80%)

To determine the volume of culture to store in your cryotube, subtract the volume of 80% glycerol to add from 2mL.

Note: We suggest storing E.coli at 16% glycerol, so add 0.4mL 80% glycerol to 1.6mL culture.

Note: Sean Sleight of Sleight Beer Lab suggests storing yeast strains at 25% glycerol, so that would involve adding 625µL 80% glycerol to 1375µL yeast culture.

Note: If your organism requires other reagents for storage and you would like to share your procedure, please email yoshi@sound.bio with a request for aliquots to be made available and with your protocol so we can share it with others.

Wipe down the laminar flow hood with cleaning ethanol and a paper towel.

With gloves, under the laminar flow hood, pipette the volume of culture determined in step 8 into its its respective cryotube for each culture.

Pipette "volume of 80% glycerol to add" determined in step 7 from your sterile 80% glycerol aliquot into each cryotube.

Close each tube and invert it 3-5 times so you see a bubble go up and down as you invert each tube.

Look over the printed table of freezer box locations attached to the front of SoundBio's -80C freezer and ensure you know where your freezer box is.

Quickly, so as to not allow the temperature to decrease significantly and samples to melt, place your organisms stored in glycerol in your freezer box and shut the -80C freezer properly.

Cleaning

Properly bleach any leftover culture before pouring down the sink.