I have a problem with adapters dimmers to perform an NGS. I would like to know how the best way to separate my library from the contaminant dimer. My library has approximately 200 bp and the dimers are at 150 bp. Could someone help me? Should I use polyacrylamide gel or agarose?
There are two main ways to remove extra adapters: gel separation and magnetic bead selection.
For gels, I always used agarose gels and had success (Invitrogen e-gels specifically). However, magnetic bead selection is MUCH easier to do. For that, you want AMPure XP beads. They are expensive, but they are worth the money.
The most important thing is to do two size selection steps: one before PCR, and one after PCR. If you only do one clean-up, you will always have too many adapters in your final library.
Andre Borges Veloso
I have a question about your insert size? It seems that you have a very small insert? What gene are you trying with illumina? I guess the trouble shooting should start from there.
Nevertheless, trimmomatic is definitely useless here as Richard Casey suggestedd earlier. To use it, first you have to generate the data, which is obviously a waste of money to sequence empty adapters.
Suggestion by Jennifer Hardee is much more practical. Although there is some confusion in the explanation of two step clean-up via magnetic beads, generral idea is correct.
Magnetic beads can seperate the primer dimer but I dont think it would be any help if you primer size is 150 and primer+insert is 200. You will loose a lot of target yet it will contain the primers.
The best option here would be gel based purification/extraction. But normal gel extraction with column based purification will/might result in very low recovery, which might not b sufficient for further analysis. In such situation E-gel suggested by Jennifer Hardee is the best option, Simple, fast with good recovery.
Dear Andre Borges Veloso ,
we are using dual beads ampure size selection in my lab for issues like this.
You will find a useful protocol here: https://research.fhcrc.org/content/dam/stripe/hahn/methods/mol_biol/SPRIselect%20User%20Guide.pdf
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