▲ CRITICAL STEP To minimize error in amplifying sgRNAs, it is important to use a high-fidelity polymerase. Other high-fidelity polymerases, such as PfuUltra II (Agilent) or Kapa HiFi (Kapa Biosystems), may be used as a substitute. ivPerform a PCR by using the following cycling conditions: Cycle numberDenatureAnnealExtend195 °C, 2 m2-3195 °C, 20 s60 °C, 20 s72 °C, 20 s3272 °C, 3 minvAfter the reaction is complete, run a sample of the product on a gel to verify successful amplification: cast a 2% (wt/vol) agarose gel in TBE buffer with SYBR Safe dye. Run 5 μl of the PCR product in the gel at 15 V cm–1 for 30 min. Successful reactions should yield a single 370-bp-long product, and the template should be invisible.
