Hi everyone!!
I have a problem with my cfDNA libraries. I'm trying to obtain a specific product with adaptors for NGS. I use a comercial kit, but when I analize the results with bioanalyzer , a inespecific and unexpected peak appears with 800bp of size (my peak of interest is about 300bp)
Do you have any idea about what is this?
My starting concentration was 6,86ng, and after 11 cycles, I obtained 410,6ng
There is an image attached that shows what I explain:
Hi Ana,
Which kit are you using? Can you say a little bit about the library construction protocol? Maybe you can check if your adapters sequence has some similarity with your gDNA.
And did you purify your library after construction? I have never analyzed my library prior purification, so I have never seen this before. And we also purify it before adapters ligation.
Hi Michele,
Thanks for your answer. The kit that I'm using has 3 steps, and I am not pretty sure about the molecular mechanism in each one, but the steps consist in a template preparation, library synthesis and finally, a library amplification without any purifications between them. I usually do a purification after the final step with beads, and then I check with bioanalyzer, but in this case, my concentration was too low and I decided to do a bioanalyzer previous to the purification in order to adjust the ratio of beads.... and I found this effect...
my thoughts are that probably there is a overamplification effect, but I'm not sure. What do you think? thanks in advance!
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