to a final concentration of 10 μM in ddH2O by adding 10 μl of the 100 μM primer stock to 90 μl of ddH2O.U6-sgRNA PCR. Set up the following reaction for each U6-Rev primer as follows: ComponentAmount (μl)Final concentrationHerculase II PCR buffer, 5×101×dNTP, 100 mM (25 mM each)0.51 mMU6 PCR template (pSpCas9(BB))10.2 ng μl−1U6-Fwd primer (universal)10.2 μMU6-Rev primer (sgRNA specific)10.2 μMHerculase II fusion polymerase0.5Distilled water36Total50
