I have performed PCR with 50 μL, which showed a clear band in the gel electrophoresis. I then performed large-scale PCR with 3650 μL, which showed an unclear band in the gel electrophoresis. What can be reason for this? Could it be plasmid loss even though there is a band, just a very unclear one? It should be mentioned that the single PCR was performed in the summer, and the large-scale was performed now
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1$\begingroup$ It could be poor mixing of the reagents, poor thermocycling of the larger volume, or a host of other issues. More details on how both experiments were conducted would be useful. Is the 3.6 mL reaction in a single tube? How are you cycling a volume that large (separate water baths)? Did you increase the length of your steps to account for slower temperature change of the larger volume? $\endgroup$– acvillDec 22, 2020 at 14:46
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1$\begingroup$ @acvill thank you. I made the large-scale in a falcon tube and then moved the mixture to PCR tubes each containing 50 μL, so the PCR conditions should be the same as the single PCR amplification $\endgroup$– F.SalDec 22, 2020 at 15:01
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1$\begingroup$ Still the question is: Did you mix really well? $\endgroup$– Chris ♦Dec 22, 2020 at 15:04
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$\begingroup$ Do you have photos of your gels? $\endgroup$– gaspanicDec 22, 2020 at 19:03
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I made the large-scale in a falcon tube and then moved the mixture to PCR tubes each containing 50 μL, so the PCR conditions should be the same as the single PCR amplification
If all reagent concentrations and cycling conditions are the same, I suspect that you did not sufficiently mix your large-volume reaction prior to dispensing as 50 μL aliquots.
It should be mentioned that the single PCR was performed in the summer, and the large-scale was performed now
If you were using the same polymerase and primer stocks for the small- and large-volume reactions, were the stocks properly stored in the interim? Recombinant DNA polymerases in glycerol buffers have shelf lives of months to years depending on the supplier specifications and storage conditions, though you should expect some decreased activity after several months.
