will the plasmid DNA be damaged during the purification process?

Question

How is the expression of the foreign gene crried by the plasmid DNA performed?

currently done an experiment on plasmid DNA extraction from recombinant E.coli just wanna know whether the plasmid DNA would be damaged during the purification process or not. cos i couldn't get the answer from the net... so i hope anyone with vast knowledge on DNA could help me out. thanks... and also, how would you perform the expression of the foreign gene carried by the plasmid DNA?

Answer 1

It depends on the method you are using.
If you follow the protocol carefully (for any method) then there is no problem. For example if you are using a kit and doing alkaline lysis don't allow the lysis to occur for more than 5min-this however is more to ensure that you don't get genomic DNA contamination.

Expression of the gene depends on the plasmid, promoter and strain you are using. Some plasmids are used only for cloning (no expression) others for low/moderate expression for in vivo studies and others for over-expression in order to isolate the protein. Some have the gene being constantly expressed, but usually (especially when you are doing over-expression), expression is inhibited by a repressor and at an appropriate time of the growth curve of the bacteria you add a small molecule(inducer) that binds to the repressor and relieves repression, leading to expression of your gene. The most common ones are IPTG and aTc (depending on the promoter).

Answer 2

A little more specifics and we could probably help you.