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Chunk #74 — PROCEDURE — Functional testing: detection of indel mutations by the SURVEYOR nuclease assay ● TIMING 5–6 h

Source
Genome engineering using the CRISPR-Cas9 system.
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■ PAUSE POINT The digested products with Stop Solution can be stored at –20 °C for at least 2 d at this point for later analysis. 84Visualizing the SURVEYOR reaction. SURVEYOR nuclease digestion products can be visualized on a 2% (wt/vol) agarose gel. For better resolution, products can be run on a 4–20% gradient polyacrylamide TBE gel. Load 10 μl of the product with the recommended loading buffer and run the gel according to the manufacturer's instructions. Typically, we run the gel until the bromophenol blue dye has migrated to the bottom of the gel. Include the DNA ladder and negative (untransfected) controls on the same gel.85Stain the gel with SYBR Gold dye diluted 1:10,000 in TBE (20 μl of stock in 200 ml of TBE). Rock the gel gently for 15 min. Be sure to shield the staining solution from light to avoid photobleaching of the dye.86Image the gel by using a quantitative imaging system without overexposing the bands. The negative controls should have only one band corresponding to the size of the PCR product, but they may have