Hi there,
The transformed cell is not stable enough in time therefore yield of production might get lower if using the same clone on and on.
Yes it is highly advisable to make it as a fresh culture!
Hi Ravi,
Always use fresh culture to study expression. Plates last for 4-5 days and they start losing the plasmid of your interest. Better you go for glycerol stock. Prepare glycerol stock of your BL21 transformed cells, store them at -80. Use each vial once for your expression study. This will improve your expression results.
In my hands it's generally fine to grow up one generation and freeze many stocks of that. Then those can be used for as many expression experiments as you care to prepare vials.
This will work less well if your protein is toxic, but in my hands that has been the exception rather than the rule. You can remedy that problem by eliminating the LB and growing the stocks in professor Studier's non-inducing media.
Quick answer is, You need not; unless you have some of the issues mentioned already (toxicity, selections of bacteria that have lost expression, etc). If you get vastly reduced protein expression or truncated proteins, then you have a case for retransforming everytime you express. Otherwise just donot bother. However, convincing a supervisor who insists on this retransformation everytime is?? I like John's suggestion on this.
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