Hello everyone,
I did a TA cloning. My DNA was 70 bp and the vector was PTZ57R/T. To check the insertion I did PCR by Plasmids extracted from white colonies and M13 (as primers). As result, I found many PCR products that are small (approximately 150 bP), even smaller than blue colonies. On the other hand, I have not any bond in control PCR. It means these very small bonds are not dimer primer. I am wondering how I can interpret this result.
Is the negative control done using the empty vectr as a template ? If you do not have any band in your negative control then it means your insert has been cloned in. But your expected amplicon size is too small to not be confused as primer dimers. Thermal diffusion usually causes the smaller bands to look broad and primer dimer sizes are not always consistent.
Since your insert is small you need to have one primer on your insert and another one on the backbone a reasonable distance away (about >400 bp away) so that the amplicon cannot be mistaken for anything but your clone.
If you are having trouble validating the clone by PCR. An alternative would be to double digest the plasmid and release the insert. But since your insert is 70bp you can take one unique enzyme in or at the start of your insert and another unique enzyme on the vector backbone about 400 to 700 bp away from the insert. This way your fallout would be bigger and you can see that an extra 70bp has been added to your insert. If the 70bp addition is hard to see, the empty vector alone can be simultaneously digested with the same enzymes to release a fallout about 70 bp less than your clone.
Dear Praveesh
Thank you for your answer. It was helpful.
My negative control included just primers and as another control, I chose one blue colony and I did PCR the blue colony.
But unfortunately, I do not access to the digestion enzyme.
I put the picture of my gel here. Maybe, it helps.
hi
1-you should get160bp PCR product by "M13/pUC sequencing primer" using colony-PCR method for blue colonies or self ligated vector .
2- you should get 230bp PCR product by "M13/pUC sequencing primer" using colony-PCR method for white colonies .
Note : To improve ligation reaction ,you should use T4-ligase fresh buffer & use 0.5 or 1 ul PEG 4000 in ligation reaction mixture ( incubate over night in 4c)
3- you can run colony-PCR with "M13/pUC sequencing primer" ( Ta=48c , ET 72c = 15 sec , PCR cycle 25 )
4- after get the true clone , you can culture recombinant E.coli in 5ml LB-Amp (100ug/ml) in loosely-cap tube ( 37c , 250 RPM , 16 hour ) then purified plasmid and check the insert by restriction enzyme digestion .
good luck
Dear Mr. Hadavi
Thank you for your advice.
I did exactly what you said except the PEG.
I can see the blue bands around 160 bp, but some white clones are very short. Even smaller than blue colonies. I do not know what they are.
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