Currently I am over-expressing the cloned gene in E.coli BL21(DE3) and harvest it and wash it with phosphate buffer. Then making a 20% cell suspension in 50 mM phosphate buffer pH 7.5 containing 50% glycerol. Aliquots that are used once or more, stored at -20 for further activity.
1. What improvements are suggested on the above storage method?
2. Can the percentage cell suspension be increased (to say 40%) so as to reduce the amount of glycerol and buffer in the reaction (less buffer from enzyme to reduce buffer interference during buffer optimization)
Snap freeze the aliquots in liquid nitrogen and store them in -70. Those samples will stay for longer time without loosing activity.
Please explain what you mean by whole cell activity? Storing cells at negative 20 Celsius is not a good idea at all. You may want to store the cells at negative 80 Celsius or below as suggested above.
Dear Mangal,
In that case all you need is to resuspende the cells in 8% glycerol and snap freeze in liquid nitrogen or dry ice/ethanol bath and store at negative 80 Celsius.
- Badges
- Science method