- 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).
- 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.
- 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).
- 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.
- 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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