I am not sure what selection/enrichment you are using to increase the proportion of 'mutant' plasmid, but usually we do see varying degree of the 'wild-type' plasmid. Pick the clone (colony) showing the desired mutation at 100%. You may have to go through a second round of transformation with dilute DNA and screening for such 'pure' mutant clones.

I amplify a particular region between two resteiction sites of different enzymes. And then digest both the plasmid and pcr product. Followed by gel run of the digested product. From the gel i elute the digested pcr product(insert) and one band(vector) of the plasmid, leaving the second band (which is same as pcr product but no mutation). Then I ligate and transform. After that when I do plasmid prep and digestion for some plasmids I see wild plasmid type pattern and for some mutant plasmid type pattern

Hi there,

So you are not confronted with mutagenesis issue but with cloning issue. If you find wt sequence at the end it means the initial plasmid hasn't been properly digested (left circular or just linearized) and has self circularized during ligation. Did you run ligation controls (vector alone in the ligation mix without insert without ligase and vector with ligase without insert)? If no you should as they will tell you where do the clones come from actually... Including non digested vector or self circularization: if clones with the control without ligase without insert then it's due to non digested vector; if clones with control without insert only then they come from either non digested plasmid or from plasmid self ligation.

Actually this question is a general question, not for the experimental issue, because as told before I do get mutated plasmid along with the wild plasmid.

But, yes I have once transformed cells with ligation mixture having only vector, but no insert. I didn't get any colonies. It means digestion was complete. But after every transformation, both wild and mutated plasmids I get. So, just I was curious to know from where it comes.

If the WT type plasmid is of E cloi origin then you should use Dpn1 digestion to get rid of the WT plasmid. Have you done it?

So you are cloning both wt and mutant within the same ligation, right? So both PCR copies are present in the ligation mix. YOur PCR product is not homogenous then...

No. I already have a plasmid with the gene of interest. What I am doing is I am introducing mutation in that gene with SOE-PCR, by amplifying only a small region present between two unique restriction sites (each site present only once in the plasmid). after pcr, I am digesting both the original plasmid and the PCR product to get sticky ends in them. after the digestion, same size of fragment is released from the plasmid as that of pcr fragment. with the help of gel extraction I am removing that original fragment and with ligation introducing the mutated fragment into the plasmid.

Since I already have PCR fragments with mutation, and I have removed the original fragment by gel extraction, then also I see some wild sequences. So, I was wondering how or from where it is coming?

one possible answer I thought was the proof reading activity of polymerase enzyme. But since mutation is in primers, enzyme can't correct that. It can proofread only what it's introducing.

OK so it might be due to the matrix you use for PCR:

Do you use the full plasmid as template? If yes do you gel purify the PCR product before digest? If not, the digest of the matrix together with the PCR product will give you some wt insert. BTW what is the frequency of wt? It should be low...

If frequency is high then it's definitely a cloning issue (the ligation control with no clone you were talking about in a previous message is vector alone with ligase or vector alone without ligase?)

Thanks Dominique, from your reply I have got the idea from where wt is coming.

Reason could be as follows-

when I do first set of PCR [PCR 1 (with primers A and B) and PCR 2 (with primers C and D)], then I use whole wild plasmid. But I purify the PCR products with PCR purification kit and not by gel extraction. So in the purified product, wild plasmids are still present.

Then I use the above PCR products further for PCR 3 with flanking primers (primers A and D). So there the flanking primers are amplifying the wild plasmids also and from there we get the wild pcr products (non mutated fragment) along with the mutated fragments (from the previous pcr products).

Thank you so much everyone for the reply.