After long time, I was doing again some PCR (if you wish to see how successfully, look here https://www.researchgate.net/post/What_is_wrong_with_my_DNA_agarose_gels-double_bands_and_weird_ladder ). During that, I was fixing the number of cycles to gain some optimal DNA amount to have nice gels. However, I have encountered somewhat weird phenomenon, which goes against my assumption.
So I'm doing PCR with the same primers, but there are different amplicons. My assumption is, that the primers should have the same efficiency, regardless of what they amplify. Is this wrong?
Well, if they produce for simplicity 1000 copies of 100 bp fragment or 1000 copies of 10 000 bp fragment, then the 10 kbp fragment contains 100-times more DNA and hence should be much thicker.
But the reality is opposite. I attach for example one recent gel, where the largest fragments ~5 kbp are very faint, while fragments somewhere around 1-3 kbp are thicker and fragments at 500 bp are just one huge blob.
What is the problem? Could the problem be with processivity of DNA Pol? Would it help to extend the elongation phase? To include the final 10 min 68°C step, which we used to do always, but now is not mentioned in any protocol?
I assume the Pol should not anneal new primers in mid of elongation phase, so the efficiency should not be much higher than 2 even for the small fragments. So does that mean that the efficiency for large fragments is much smaller?
Primers and pcr amplification do not have the same efficiency regardless of sequence. Smaller amplimers will amplify better because they melt easier and more efficiently and the enzyme does not have time to fall off the short extension needed. High GC sequences melt badly and often amplify badly as a result of this.
Long amplimers often form secondary structure and the enzyme extends a bit then falls off and a new enzyme anneals and completes the amplification but this takes time and often leads to failed amplification even if we increase the extension time. Often a longer amplimer will look stronger on an agarose gel but this is because the longer amplimer will take up more fluorescent dye molecules than a short one so looks brighter.. Very short amplimers (50bases) can look extremely faint. Below 1000 bases generally there is no need to extend the extention time but above 1000 bases using 1minute per 1000 bases is commonly used but check what the enzyme information sheet says.
I would expect the 5kb amplimer to be improved (stronger) with a 5 minute extension time.
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