I´m working with a "homemade" Tn5 transposase and I want to cut my ds cDNA after pre-amplification with a single cell protocol (Smart-seq2). My library has TONS of primer dimers and concatemers in the range 50-170 bp and I can´t get rid of them just decreasing the ratio Ampure beads:DNA. I was wondering if these short fragments are usable by the Tn5 or are too short. If they are not used the problem is solved, but if they can "tagmented" my library will have 90% of reads consisting of primer dimers! Any help is appreciated, thanks!
Hi, I once ran into a similar problem, primers concatemers (must have been >2, each primer was ca. 35 bases long) of 100 bp, regardless of the substrate. I couldn't find a way to get rid of this background either by cleaning up the product, but it eventually got resolved by starting the pre-amplification in a pre-heated cycler, rather than a cold block. So far for using hot-start polymerase... I used Kapa high-fidelity master mix.
In conclusion, the problem might be better solved by changing the PCR conditions rather than trying to remove it afterwards.
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