Yeast or bacterial biomass from a range of sources can be mixed with glycerol to final concentration of ~25 % (v/v) and frozen indefinitely at -80°C.

The simplest way to freeze a strain is to inoculate a plate of solid medium and incubate it for one or two days at the optimal temperature of the strain. This medium has to be selective if the strain requires it.

Prepare as many screw cap tubes tubes with half of the maximum volume with an appropriate liquid medium or sterile water.

If you are freezing yeast strains, YPD or some other rich medium can be used even if the strains have plasmids. For E. coli LB medium can be used without antibiotics even is the strains carry plasmids. Transfer cells from the solid medium to the tubes using a large flat toothpick. Take as much cells as you can.

Add one volume of 50% glycerol to each tube so that the final concentration is 25% (v/v).

Resuspend cells by vortexing and let the tubes stand in room temperature for an hour up to a day before freezing. This is for the glycerol to have time to enter the cells before freezing which may improve the survival of the cells.

Alternatively, grow strains in >1 ml medium overnight at the appropriate temperature with shaking. Fill about half of the maximum volume of the freezer tube. Add 1 vol of 50% glycerol. Mix well.

Put the tubes in the -80 freezer and add the position and box name into list kept in the Google spreadsheet that we share within the group.

Adapted from this.

Ideally, at least three to four different successful clones of each new construct are identified at the end of a construction project. In the case of new plasmids, one or two clones are sent for sequencing. We do not freeze all positive clones due to freezer space limitation. Instead, we freeze two tubes, the most convincing/most studied clone alone in one tube and another tube containing a pooled mixture of all positive clones, including the first clone. The second tube should be marked "pool" or "mix".

All further work should be done with the first clone. The pooled clone is useful if at some point further clones need to be studied.

Prepare an ice box with ice, sterile toothpicks, solid medium and somewhere to work before removing the tubes from the freezer.

Open the tube near a flame and scrape off some of the solid cells with a sterile toothpick. Transfer the cells to solid medium and return the tube to the freezer as soon as possible and without ever letting the culture melt.

Reference: Sherman, F, et al. (1986) The laboratory course manual for methods in yeast genetics. Cold spring harbor press, cold spring harber, NY, P179.