I have a standard pUC19 plasmid that I would like to clone GFP, a promoter, and a new ORI into via restriction free cloning. However, I have some concerns about using the original sequence.
On account of the high specificity of restriction free cloning, I'm concerned that the introns of the plasmid (where I plan to insert them) may have mutated since pUC19 is relatively old and widely used.
Is this a valid concern, or should I proceed and not worry about it?
Hi there,
pUC19 is a commercial plasmid which sequence has been established and therefore it is guaranteed when you purchase it. Why do you have doubt about it? You could easily sequence it yourself if you are not confident enough.
I agree with Dominique, sequence your plasmid, it will tell you if there are mutations.
To my knowledge, the two ORFs of pUC19 are the beta-lactamase gene and the lacZ alpha gene, which contains the multiple cloning site. Neither contains introns. Do you mean non-translated (i.e. intergenic) regions ? Otherwise, I agree with Dominique and Cyrille.
Thank you all for your suggestions, I appreciate it.
I think I will run the RF reaction, then a gel to see if it worked. I'll have it sequenced if not.
Yes, sorry, that's correct, I meant the intergenic region between the ori and lacZa.
Thank again.
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