I am doing droplet based digital PCR inside plastic chips. I can get the correct band, but I also consistently get an unexpected band, slightly shorter. I attached a typical gel image here. Column1 is the ladder. Column2 is the no template control in a PCR tube. Column3 is the positive control in a PCR tube (the band is ~ 100bp). Column4 is from droplet PCR, I collect drops from the chip, break drops, run the gel. I have two bands near 100bp. I suspect there is some contamination from my chip, but I would like to get some suggestions from you guys about what is the possible contaminations? Any additives I can try to get rid of the contaminations?
I do not know is the simple answer but are you sure that this is contamination?. It may not be possible to compare tube and chip directly because the temperature profiles may be different. If the chip cools quicker then you may just have more primer dimer and annealing to a different part of the genome at the lower annealing temperature to give the second band. Sequencing it may point to the answer. I assume that the negative control of a pcr in a chip without template dna gives either no amplification or just primer dimer
I see the smear band above, the PCV condition or the primers maybe not good.
it works for the pure positive control, but not good/specific enough for the samples.
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