I just used your CleanTag Ligation Kit for Small RNA and I am trying to determine if the ligation/library prep was successful. Am I correct in assuming that I expect to see essentially no adaptor dimer formation in the prep? The TapeStation results show large (~2-4 ng/µl) peaks at around ~140bp for each sample (magnetic bead cleaned) and no obvious peaks at what would be the adaptor-dimer (~120bp). I just wanted to confirm that because of the CleanTag chemistry, no adaptor-dimers form during ligation, and therefore, they aren’t amplified. Is this correct? If true, I should also be able to load these preps straight onto a sequencer with no gel purification of the 140bp band? – Steve

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Dear Steve,

You are correct in that the CleanTag™ chemistry can fully suppress adapter dimer formation. If you are using very low input levels (less than 10 ng) you may start to see some adapter dimer but it should be minimal compared to amount of tagged library. Dilution of the adapters per the product insert is key to keep adapter dimer low at lower inputs.

Yes, you can load magnetic bead purified samples without gel purification directly onto a sequencer and get good quality sequencing data.

Best regards,
Sabrina Shore, MS
Scientist

2 thoughts on “I just used your CleanTag Ligation Kit for Small RNA and I am trying to determine if the ligation/library prep was successful. Am I correct in assuming that I expect to see essentially no adaptor dimer formation in the prep? The TapeStation results show large (~2-4 ng/µl) peaks at around ~140bp for each sample (magnetic bead cleaned) and no obvious peaks at what would be the adaptor-dimer (~120bp). I just wanted to confirm that because of the CleanTag chemistry, no adaptor-dimers form during ligation, and therefore, they aren’t amplified. Is this correct? If true, I should also be able to load these preps straight onto a sequencer with no gel purification of the 140bp band? – Steve”

  1. A follow-up question- I’ve successfully made small RNA libraries using this approach, but my most recent prep has a second peak at around 90-110bp (in addition to the expected146bp). Any idea what this is? Too short to be adapter dimers, correct (~125bp)?

    I have about 100-200 ng total RNA input, using 1:2 adapter dilutions and 15 PCR cycles. I can’t figure out what the ~100bp peaks would be?

    • Dear Steve,

      We often see a peak at ~100-110 bp. We know it isn’t specific to our kit however we don’t know exactly what the band is. We speculate it could be RNA concatemers with perhaps one of the adapters ligated on. It most likely does not contain both adapters so it should not amplify well on the flow cell. Please contact us if you have any additional questions.

      Best Regards,
      Sabrina

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