▲ CRITICAL STEP SURVEYOR assays rely on the detection of single-base mismatches; therefore, it is crucial to use a high-fidelity polymerase. Other high-fidelity polymerases, such as PfuUltra (Agilent) or Kapa HiFi (Kapa Biosystems), may be used as a substitute. In addition, because SURVEYOR cleavage results can detect naturally occurring single-nucleotide polymorphisms, it is important to run negative control samples of untransfected or otherwise unmodified cells. 76Perform a PCR with the following cycling conditions, for no more than 30 amplification cycles: Cycle numberDenatureAnnealExtend195 °C, 2 min2-3195 °C, 20 s60 °C, 20 s72 °C, 30 s3272 °C, 3 min77Run 2–5 μl of the PCR products on a 1% (wt/vol) agarose gel to check for single-band products. Although these PCR conditions are designed to work with most pairs of SURVEYOR primers, some primers may need additional optimization by adjusting the template concentration, MgCl2 concentration and/or the annealing temperature.
