1. Cast a 150 ml 3% agarose gel with 1X TAE buffer and 400 ng/ml EtBr (60 μg) using a gel comb that can accommodate approximately 60 μl in each well. Recommended well size: 1 mm (length) x 8 mm (width) x 7 mm (height).
  1. Mix 30 µl of each PCR product from the Step 6 amplification with 3 µl of 10X loading buffer (e.g. 50% sucrose with Bromophenol Blue tracking dye) and load onto the gel. In at least 1 well, include a 100-bp DNA ladder.
  1. Run gel at 5 V/cm until the 100 bp band is about an inch from the bottom (usually about 1 hour), then using a UV Transilluminator and clean scalpel, excise a 2 mm narrow band from the brightest region in the 350-450 bp size range (see Example Gel Data image).

LINE-1 Mobilization Assay Example Gel Photo
Example Gel Data

  1. Isolate each PCR product from the gel fragment using the QIAquick Gel Extraction kit and following the manufacturer’s protocol. Elute in 30 μl of QIAGEN EB Buffer.
  1. Measure concentration of each purified P7-Index-3’L1H-DNA-P5 product using a NanoDrop 2000 (or similar spectrophotometer) at OD260. Combine different Index-tagged DNA samples together at equimolar amounts into the final sequencing sample pool. Dilute the combined DNA samples to 10nM (approximately 2 ng/µl).
Last modified: Jan 05, 2017

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