Hi guys,
a situation has just come up. I want to integrate a restriction site into a Plasmid (~6.5 kb) by PCR mutagenesis. After PCR (25 cycles), I observed the DNA band on the gel, however very weak and right below another band could be seen. I extracted the DNA band of proper size and inserted into E. coli by Transformation twice. I picked 5 colonies from each LB-Amp plate and isolated the plasmid. My theory was that if the restriction site is integrated succesfully a double Digest using the just integrated and a present restriction site should lead to two fragments of known size. I did that. Unfortunately, I do not get two fragments but only linearization. The ristriction site should be integrated by PCR. So why did it not integrate? Do I have to Isolate the plasmid of all clones I have on the plates (~ 12 on each plate) but this normally is done for Screening of inserts in a vector ? I am using the proof-reading Vent DNA Pol for in PCR. Is it possible that the Enzyme recognizes the looped restriction site and deletes it ?
I am very happy for any Suggestion !! Thanks in advance
Hi Stefan,
Did you treat your PCR reaction with DpnI after your mutagenic PCR ? DpnI only attacks methylated DNA (in your case the in vivo grown wild type DNA that you used as a template), which will reduce your background of wild type clones considerably.
Good luck
Pierre
Do you have a good positive control for the enzyme digestion that allows to exclude low efficiency of these reactions?
Anyway, PCR mutagenesis efficiency can vary widely. DpnI digestion favors the isolation of mutated clones as explained above, but even doing DpnI digestion on mutagenesis products, you get a mixture of mutated and non-mutated clones, and even some clones containing both mutated and unmutated plasmids. You can analyze more clones, of course, and even do electroporations with the mutagenesis products in order to get higher numbers of colonies to screen. Sequencing screening would give you the final answer not only about the insertion of the restriction site, but also about the overlapping of mutated and non-mutated sequences in a given clone, and about the occurrence of other undesired mutations in the neighborhood of your targeted region. I would prefer to screen by directly sequencing.
If after screening more clones, you don't get the desired mutated plasmids, it would be good to recheck the design of mutagenic oligonucleotides, as low efficiency mutagenesis tends to be related to primer design. For instance, its good to have G/C-rich sequences at both end of mutagenic oligonucleotides. Primers forming dimers or loops that are stable at temperatures close to the one used for annealing should be avoided.
I do not extract mutagenesis products from a gel, I just transform cells with them and sequence the resulting clones.
Thanks to your answers. I think I will sequence the plasmids and see what´s going on.
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