Hello, good evening. I have a question that arose when running an agarose gel for DNA. Some time ago, we transfected CHO cells with a plasmid and wanted to see if it could transcribe with that plasmid, we used that RNA to make some cDNA. Lane 1 indicates the marker, the second indicates the control of the PCR reaction for the plasmid, the third indicates CHO cells without the plasmid, the fourth lane indicates the circular plasmid, the fifth indicates the linearized plasmid, and the sixth indicates the pure vector. So far, so good. The problem is with the Beta-actin controls. When running them in the same order (except for using another cell line as a control), the lane corresponding to the circular plasmid (ninth lane) shows an extra band. At first, I thought it was some kind of primer non-specificity for Beta-actin, but the problem is that they did not amplify the same corresponding band for the same linearized plasmid (tenth lane). It couldn't be contamination in the reaction because negative controls and controls without the plasmid did not amplify. It also couldn't be contamination of the master mix because if so, all the lanes would have amplified. The DNA quantities vary by 1-4 nanograms and have similar degrees of purity. I don't know what could be happening since the same linear plasmid did not amplify, but it did for the circular one for Beta-actin. I have carried out the experiment twice and will do it again, but I have not found the reason why that band could have appeared.
I placed some arrows so you can identify the band that correspond the cdna of the plasmid transcript (red ones) and the unknown band (green one), and yes, they have similar sizes but not the same.
i also add another gel for only the B-actin PCRs of that same cDNA (the third lane correspond to the same double band pattern)
To me it looks like non-specific amplification. The primer might be binding to some non-specific region (in addition to the gene of interest) of the circular plasmid which might not be happening with the linear plasmid.
Have you done real time PCRs for these? and do you have met curves corresponding to these samples, if yes then that might give us better idea.
Also, what samples did you run on lane 8?
A faint band corresponding to the non-specific one, can be seen in sample run on lane 8.
That 8th lane is the CHO cells that weren't transfected (B-actin). May I ask why this could happen with circular plasmids but not with linear plasmids? They are both the same, the only difference is linearization.
Not sure what exactly might be the reason. Mostly has to do with structural differences between the two.
You can go through the following articles which might give you a better idea.
1) Discussion section of
https://www.frontiersin.org/articles/10.3389/fmolb.2020.00155/full
2) Article Serious Overestimation in Quantitative PCR by Circular (Supe...
Try increasing the template concentration of the circular plasmid and see if you are getting non-specific amplification.
Oh, i will check then, because, that's just the product of cDNA, so it would be rare to get that, because i didn't extract the plasmid.
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