I'm using the pET-46 Ek/LIC vector to transform e.coli NovaBlue in particular. I've had no problems generating my 1600bp insert, and annealing it to the vector. The sequence of the insert checks out just fine, and I go ahead and transform NovaBlue cells with the annealed insert + vector.
When I plate out the cells on antibiotic containing agar I get many many colonies. However when I do colony PCR and sequence the amplified insert, the insert always has mutations. I have not been able to find one yet that does not have at least two non synonymous site (amino acid changing) mutations. Is this typical? How many colonies do I need to screen, on average, to find one without non-synonymous-site mutations?
why do you send the colony-PCR product for sequencing? grow the colonies in broth, extract plasmid and send the plasmid for sequencing. by PCR you will end up with more mutation. this is wrong to me.