Friday, September 17, 2010

Nextera libraries from buccal-cell DNA

We’ve received several inquiries regarding the use of Nextera™ technology to prepare next-generation sequencing libraries from buccal-cell DNA. The BuccalAmp™ DNA Extraction Kit is designed for a quick extraction of DNA for PCR only. However, our R&D scientists have developed a protocol, outlined below, to ensure successful preparation of Nextera libraries from DNA extracted with the BuccalAmp Kit. For this example, DNA was extracted from four buccal swabs and pooled.
  1. Centrifuge the tube briefly (1 minute at 3,000 x g) to remove the solid cellular debris.
  2. Remove the supernatant and precipitate the DNA with 1/10 volume of sodium acetate (3.0 M) and 2 volumes of ethanol.
  3. Resuspend the pellet in 100 µl of T10E1 buffer. Centrifuge briefly to remove insoluble material and transfer the supernatant to a DNA Clean & Concentrator-5 column (Zymo).
  4. Add 300 µl of Binding Buffer and follow the manufacturer’s protocol.
  5. Elute the DNA with two aliquots (10 µl each) of T10E1 buffer. Use 50 ng of eluted DNA, as determined spectrophotometrically, in the standard Nextera protocol.

Human buccal DNA after tagmentation reactions with the Nextera™ DNA Sample Prep Kit (Illumina-Compatible).  DNA was extracted and cleaned up as described above, and treated as follows: lane 1, no treatment ; lane 2, Hind III digestion; lane 3, Nextera tagmentation with HMW buffer; lane 4, Nextera tagmentation with LMW buffer. Lane M, 100-bp DNA ladder.

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