Chances of Mutation - (Nov/03/2006 )

I want to ask after ligation and transformation into E.coli is there any probability of mutation inside bacteria or while the chances of mutation is during PCR reaction.
Mutation can happen when you are transforming your clone for expression into expression host or
if you are sure that this construct is getting express and used it for many times in E.coli for amount amplification mutaion can be happened or not,
regards
samita

I'm not certain what you're saying in the second half?

but, I can tell you that it depends on the nature of the mutation and also whether your e coli host is RecA+ or RecA-

My second Question is

I have sequene one clone its in reading frame and all the base pair are compleletly match with my Original sequece when i sequence it. Now i am trying to express it in Expression host.
But every time i need fresh transformation so i again transform it in Dh5 Alpha and then i have enough amount of my constrcu that i can every time have fresh transformation in BL21 DE3 cell so tell me during this transformation there are chances of mutaion or not.
regards

why do you re-transform every time?

why can't you transform in to BL21(DE3), grow some up and make freezer stock, then use the freezer stock for fresh cultures to do your expression experiments? it should not be necessary to transform through two strains every time you want to express your protein