Difference between revisions of "Glycerol stock"
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[[Synchronized Competent Yeast Cells]] should be frozen in [[Lithium Acetate with glycerol|Lithium acetate + 30% Glycerol]] as this increases their transformation efficiency ([http://dx.doi.org/10.1002/yea.1247 Suga & Hatakeyama, 2005, doi:10.1002/yea.1247]). | [[Synchronized Competent Yeast Cells]] should be frozen in [[Lithium Acetate with glycerol|Lithium acetate + 30% Glycerol]] as this increases their transformation efficiency ([http://dx.doi.org/10.1002/yea.1247 Suga & Hatakeyama, 2005, doi:10.1002/yea.1247]). | ||
+ | |||
+ | === Evolution experiment === | ||
+ | For the evolution experiment the generations are frozen in 96 well flat bottom plates. | ||
+ | * Spin down cells in the deepwell plates at 1500xg for 3 min. | ||
+ | * Remove supernatant with a 300µl pipette | ||
+ | * add 150µl YES+15%glycerol | ||
+ | * resuspend all cells by pipetting up and down | ||
+ | * with the same tips, move the content to a fresh flat-bottom plate. '''Make sure you do not touch the side of the wells to avoid cross contamination when opening the plate in the future.''' | ||
+ | * carefully stick a plastic adhesive film onto the plate. | ||
+ | * replace the lid | ||
+ | * carefully without getting the plastic film contaminated, place the plate in a box and freeze in the -80C freezer | ||
== ''E. coli'' == | == ''E. coli'' == |
Revision as of 16:06, 24 February 2020
Pombe
- Grow cells overnight in 5ml liquid medium shaking.
- Spin down the cells (or leave overnight in the fridge)
- Remove supernatant
- Resuspend in YES + 15% Glycerol
- Transfer the cells to a 1.5ml (cryogen)tube
- Immediately freeze at -80°C
- register the tube in the lists
- alternatively:
- transfer 0.5ml of liquid culture to a fresh tube
- add 0.5ml 30% or 216 μl 50% sterile Glycerol to the tube
- mix, store and register
Synchronized Competent Yeast Cells should be frozen in Lithium acetate + 30% Glycerol as this increases their transformation efficiency (Suga & Hatakeyama, 2005, doi:10.1002/yea.1247).
Evolution experiment
For the evolution experiment the generations are frozen in 96 well flat bottom plates.
- Spin down cells in the deepwell plates at 1500xg for 3 min.
- Remove supernatant with a 300µl pipette
- add 150µl YES+15%glycerol
- resuspend all cells by pipetting up and down
- with the same tips, move the content to a fresh flat-bottom plate. Make sure you do not touch the side of the wells to avoid cross contamination when opening the plate in the future.
- carefully stick a plastic adhesive film onto the plate.
- replace the lid
- carefully without getting the plastic film contaminated, place the plate in a box and freeze in the -80C freezer