1260 infinity multi detector system

Stoichiometry determination of the supercomplex was performed using size-exclusion chromatography coupled to dual-angle laser light scattering. Supercomplex was first purified using Ni-NTA affinity chromatography followed by ion-exchange chromatography on a Heparin column. The purified fraction was complexed with a pre-spacer DNA and an sgRNA having sequence complementarity to the pre-spacer DNA being added. The complex was then injected into Superdex 200 10/300 Increase coupled to an Agilent 1260 Infinity Multi-Detector system. The buffer composition was 20 mM HEPES, 300 mM NaCl, 1 mM TCEP, 1 mM EDTA, 5% glycerol at a pH of 7.4. A 658 nm laser was used to perform light scattering. UV (280 nm), UV (260 nm) and light scattering signals at 90° were collected. The eluting fractions were analyzed to assess the molecular weights of the components. Bio-SEC software (Agilent) was used for data analysis and molecular mass calculations.

An Agilent BioSEC Advance 300 Å, 4.6 × 300 mm or GE Superdex 200 5 15 column was used for analytical SEC and SEC-SLS in 20 mM tris(hydroxymethyl)aminomethane-HCl pH 7.4, 150 mM NaCl buffer. Both were performed on an Agilent 1260 Infinity Multi-Detector System using refractive index to calculate masses.