Friday, June 18, 2010

Get rid of the small stuff

We have received quite a few enquiries from our customers on how to remove low molecular-weight (MW) fragments from the Nextera™ library following tagmentation and limited-cycle PCR. Based on extensive R&D testing, we’ve found that AMPure® XP beads from Beckman Coulter work very well in removing fragments below 350 bp. The 0.7X bead concentration is effective at removing lower MW fragments (<350 bp) from Nextera libraries.

Note: The 350-bp cut-off point refers to fragments below 350 bp; the actual insert size (of the genomic DNA) is approximately 250 bp (Nextera Roche-Compatible) or 215 bp (Nextera Illumina-Compatible). The reason for the difference is due to the adaptor sequences on the genomic DNA fragments. The Roche library fragments contain 98 bp of adaptor/transposon-end sequences, and the Illumina library fragments contain 135 bp of adaptor/transposon-end sequences. These additional adaptor/transposon-end sequences make the genomic DNA fragments appear bigger than their actual size.

Below we show two Bioanalyzer traces of Nextera libraries, one purified using Zymo DNA Clean and Concentrator™, the other using AMPure XP beads from Beckman Coulter.

Note: Zymo or AMPure clean-up was performed after limited-cycle PCR, just prior to loading the libraries on emulsion PCR or bridge PCR.

(click to enlarge)
Red: Illumina-compatible Nextera library, 50 ng Lambda DNA, HMW Buffer, 9 cycles PCR, Zymo DNA Clean & Concentrator-5.
Blue: Illumina-compatible Nextera library, 50 ng Lambda DNA, HMW Buffer, 9 cycles PCR, Agencourt AMPure XP beads (0.7X).


(click to enlarge)
Red: Roche-compatible Nextera library, 50 ng Lambda DNA, HMW Buffer, 15 cycles PCR, Zymo DNA Clean & Concentrator-5.
Blue: Roche-compatible Nextera library, 50 ng Lambda DNA, HMW Buffer, 15 cycles PCR, Agencourt AMPure XP beads (0.7X).

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