Hi Saheem,

EcoRI site is GAATTC

XmaI site is CCCGGG

MfeI site is CAATTG

AgeI site is ACCGGT

correct me if I'm wrong, but I see two issues here. Your PCR product has the restriction sites MfeI and AgeI engineered onto the ends to aid in cloning into the MCS of your vector. The problem, is that MfeI and EcoRI do not have the same recognition site, and are therefore not compatible "sticky" ends for cloning. Likewise, XmaI and AgeI, after digestion, do not yield compatible ends.

The result of your 3Kb product being cut down to 2.2kb and 800bp is most likely because on of those enzymes cuts your product internally and not just at the termini where you designed your restriction sites. Ligating your 3kb pcr product into the Topo vector and subsequent restriction digestion further confirms that you have an internal restriction site in your gene.

Regards,

Barry

Thanks Barry. But my gene of interest do not have Mfe I and Age I sites internally, I have checked it many times, only it is present at the ends of 5' and 3'. This is the reason we chose those enzymes to add using PCR. Secondly, I cannot add EcorI and Xma I because these sites are present within the gene. I am adding thse RS because they are the isoschizomers, and hence they will produce same sticky ends.. therefore during ligation there would be no problem. My probles is that.. though I am getting 3 kb band after performing PCR which is expected size of the gene.. but when Iam doing digestion of amplifieid PCR product (supposed to have mfe I and age I at the ends) with Mfe I and Age I am nt getting the same.

Ok i understand now. Just to clarify, those enzymes aren't considered isoschizomers of each other because they do not recognize the same 6bp sequence. Yes, XmaI and AgeI result in the same 4bp overhang but they will not cut at the same spot, because of their different recognition sequence. They should, however ligate with no problem.

Some thoughts as to what might be going on with the digestion of your pcr product.

Are you certain that your insert doesn't have PCR generated mutations that could result in the generation of an XmaI or an AgeI site? Obviously the use of a proofreading enzyme for PCR greatly reduces the chances of this.

Are you doing a double digest and if so, are you using compatible buffers? Sometimes you can get star activity of an enzyme if it isn't being used in its appropriate buffer (common during double digests with enzymes that have varying pH and/or glycerol requirements). More times than not, i have to do one digestion, ethanol precipitate or gel purify, and then do the second digest to (a) reduce the chances of star activity and (b) to get sufficient digestion of my cloning ends.

Hope this helps,

Good Luck.

Barry

Barry: I do double digestion because they are compatible in same buffer 4 of NEB. But as per your suggestion, I will also do the sequential one just to make myself sure regarding star activity..

Thank you once again.

Saheem

Saheem- your cloning strategy is sound, but your digestion results speak for themselves. You must have an internal MfeI or AgeI site present of which you are not aware. What is the source of your template DNA? Have you considered the possibility that your template has a SNP that introduces an Age or MfeI site?

You can start by troubleshooting this problem by sequencing your template DNA, and performing a control digestion on the template with these two enzymes (singly, so that you can test which one is causing an internal cleavage).