GRNA Production

1. Oligo Rehydration

100 μmol [ ] (stock) of oligos Ex. 100 μmol [ ] —> 27.8 nmol [ ] —> rehydrate oligo bead with 278 μl of H2O

• add H2O to hydrate oligos
• incubate 5 min at room temp
• mix
• incubate another 5 min to fully dissolve store at -20°C

1. Sense / Antisense Oligo Annealation

TRIS Buffer

• sufficient to allow oligos to anneal
• proper pH (7.5-8)
• 10 mmol TRIS

2 μl sense oligo, 2 μl antisense oligo, 46 μl TRIS buffer (10 mmol) ————————— 50 μl final vol

• incubate samples at high temp to low temp (about -1°C / sec) - must reach 95°C
• store at 4°C