Library preparation (amplicons arround 100 bp)

[imendez]

Hello everyone!

I am trying to prepare a library composed of amplicons of around 100 bp. After the PCR I checked the presence of amplification and then I did a cleaning step using AMPure XP beads. After this cleaning step, I lost practically all my amplicons. I tried to increase the concentration of beads (from 1.5X to 2.5X) but the result is exactly the same... Anybody know what is happening?

Thanks!

Inaki

[nucacidhunter]

It is either reagent issue or protocol. 1.5x bead would cut fragments below 100 bp. Your 100 bp amplicons should be at least 230 bp with adapters.

[imendez]

Hello,

No, I am talking about a cleaning after the PCR, before barcodes.

Thanks

[nucacidhunter]

I thought you have done PCR with fusion primers. Columns are the best option for cleaning small amplicons.

[imendez]

Thanks so much for the answer.
Do you have any recommendation?

[nucacidhunter]

Any PCR clean up column will do it. Depending on sample number you can use single columns or plate format.

[jdk787]

How did you check for amplification before your cleanup?
If you just quantified your sample you might just be reading leftover dNTPs and primers (NanoDrop), or primer dimers (Qubit) which would be lost after your cleanup.

[imendez]

I previously checked the absence of secondary structures by dissociation curves. At the same time I did Bioanalyzer and Qubit to measure the concentration...