Gel loading dye

The affinity-purified starting material and empty and vector capsid reference standards, recovered after sv-AUC analyses, were subjected to alkaline agarose gel electrophoresis. The 6 × gel loading dye (New England Biolabs, Ipswich, MA), the running buffer, and the gel casting buffer were adjusted to alkaline pH by adding sodium hydroxide stock solution to the final concentration of up to 150 mM. Thirty microliters of 1 × samples in gel loading dye and 10 μL of Quick-Load^® 1 kb DNA ladder (New England Biolabs, Ipswich, MA) in 1 × alkaline gel loading buffer were loaded on the 0.7% alkaline agarose gel. The gel was run at 3.5 V/cm for extended hours until the dye had migrated ∼2/3 length of the gel. Post-electrophoresis run, the gel was washed with 0.5 M Tris-HCl, pH 8 buffer, for 30 min. Later, the gel was incubated with staining buffer (1 × SYBR™ Safe stain in TE buffer, pH 8) for 1 h, followed by imaging using the ChemiDoc Imager.

Unless stated otherwise, a final concentration of 1 μM Cas1, 1 μM Cas2, 1 mM DTT and 10 mM MgCl₂ was incubated in reaction buffer (20 mM Tris, 100 mM KCl, 5% glycerol, pH 7.5) at 4°C for 1 h. 20 nM of radiolabed pre-spacer or 100 nM of unlabeled pre-spacer was added to the reactions and incubated at 4°C for an additional 15 min. Finally, plasmid or linear DNA was added to a final concentration of 5 nM for pCR7, or 100 nM for minimal CRISPR substrate, and the reaction was incubated at 70°C for 1 h. Reactions with pCR7 were quenched with EDTA and proteinase K (Life Technologies). Products were mixed with gel loading dye (purple, NEB) and separated on 1% agarose gels in 1× TAE. Reactions with minimal CRISPR substrates were quenched with an equal volume of Gel Loading Buffer II (Thermofisher) and 25 mM EDTA. Samples were boiled for 5 min before separation by 12% denaturing 7 M urea-containing polyacrylamide gel electrophoresis in 1× TBE. Gels were dried and radioactivity was detected with phosphorimaging (Storm 840 Scanner GE Healthcare).