Attached is the result of our qPCR, we did a library by using the ddRAD protocol. After pooling the samples from four individuals, we excised the library of 250-400 bp range by using ultra-pure agarose gel (1%) and purified it. We checked the concentration in bio analyzer and after that we did qPCR. From bio analyzer results we did not found any other peaks than the one we were interested but after qPCR when we run the gel we found some bands below 150 and above 100 bp. My question is that when bio-analyzer not showed the unimportant bands in our case, why after qPCR we have these bands, and secondly how to improve the library to avoid this issue.
Are you adding the adapters after digestion or after library excision? Are you using primers designed against the adapters? Are these primers validated for qPCR? They could be amplifying unspecifically or even forming primer dimers.
@Andre, we used adapters after digestion, we used the primers designed against the adapters and are validated for qPCR.
Have you checked:
- How is your qPCR efficiency? Large amplicons usually have low efficiency.
- Are you using SYBR? How is the melting curve? Do you have a single peak?
- Have you tried performing a control experiment in which you amplify your fragments without adapter ligation? The primers may be annealing internally to your fragments generating smaller fragments.
- Do you have the primer sequence? Try blasting it agains the human genome.
- Can you try validating your PCR (in a normal thermocycler) to check an ideal temperature? If your primers are mismatching maybe you need a higher temperature to correct it.
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