I have read about the fragile nature of transformed BL21 cells for expression studies. I have been using the transformed BL21 cells stored at normal refrigerator temperature for expression studies. According to some lab manuals it is a harmful method of storing the cells as storage temperature is quite high. Do I need to store the transformed cells at -20 degrees or -70 degrees as glycerol stock before setting up expression studies in small aliquots? Will it enhance the expression level of my gene?
It is not good to store the transformed cells for more than 4-5 days. If you want to repetedly study the expression from the same lot of cells, grow the culture from the transformed cells in LB medium to reach a OD of 0.6-0.8. Later add glycerol to a final concentration of 10% and aliquot 1ml each into sterile vials and store at -80 deg C. Use one vial each time for expression studies. This way you will get consisentely similar expession level.
It is not good to store the transformed cells for more than 4-5 days. If you want to repetedly study the expression from the same lot of cells, grow the culture from the transformed cells in LB medium to reach a OD of 0.6-0.8. Later add glycerol to a final concentration of 10% and aliquot 1ml each into sterile vials and store at -80 deg C. Use one vial each time for expression studies. This way you will get consisentely similar expession level.
Hi...
In a normal refrigerator it also depends in which medium you are storing your cells. If it's nutrient agar plate with selection marker then the shelf life can be upto half a month without any problem. If it's broth then hardly 5 days at max, as answered earlier.
Also, it is usually recommended that a fresh transformation should be performed for an expression host as the glycerol stocks of expression host tend to lose their expression levels in long term storage.
Hope this helps...
Good luck... :)
Hi,
I just wanted add one more point here. I have seen few recombinant glycerol stocks which are stable for several years. But few are loosing the insert on storage at -70 deg. within few weeks. I think it is based the insert i.e. the gene or the gene product is toxic , then it tend to loose. I wish your gene is not of that quality.
Wish you good luck.
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