It is highly recommended to check the sequence of your positive clone to make sure that no error occurred in the cloning process, particularly if your insert originates from a PCR amplification (use of a high fidelity polymerase, such as Phusion®, is advisable). Once the sequence is validated, you can transform your plasmid into the BL21 production strain. Most of the time, you won't have to worry about further mutation events, since the gene will be replicated by the proofreading DNA polymerase of E. coli. However, you should make sure that the expression of your gene is strictly repressed in the abssence of inducer, lest you should select for low producers that avoid the burden of producing a protein that they don't need and may even be toxic.

The mutation rate is entirely dependent on your choice of polymerase. It should be very, very low with a quality enzyme. That being said, you should always sequence the plasmid in your colony of choice before you express the protein.

I don't understand the rest of your question "proceed for BL21".

Agree with @Pierre Béguin

Thank you Dr Beguin. I will try to remake the insert and will take care of the points as mentioned.

Dr Katie, I mean to ask how many clones do you select to transform in BL21 once you achieve the positive fallouts. Also, do you chose multiple colonies from each transformed plate in proceed for IPTG induction screening.

It's a bit late but perhaps this will help others...

There is definitely a possibility that after you get the clone, in DH5 alfa, for instance, and get it sequenced, it will be mutated upon transformation in a different host (BL21(DE3), per your example). That is why in GMP-compliant industrial settings it is absolutely required to sequence the construct after preparing master and working cell banks. However, the probability of such an event is very low, to the point that I've never seen anybody in a research lab subjecting transformed expression strains to sequence verification, and have never witnessed such a mutation. I mean, it is remote enough that you should never use it as a possible cause when analyzing a failed experiment.

As for the second question (how many colonies to screen after transformation in BL21(DE3)?) in my experience, if you work with freshly transformed plates (that is, plates that have never been stored at 4*C and are taken straight from the incubator after a growth period of no more than 12 h), there will be no differences in expression between different colonies.

Hope it helps!

Thank you Dr. Alejandro martin for sharing your experience. I agree with your opinion and so I have already started to clone my construct with the parallel sequencing along. Hoping, this time to get the frame in order.

Thanks again.