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

(2) 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

(3) Plasmid Digestion

Plasmid DR274 - digest with restriction enzyme BsaI

Always mix then centrifuge reaction components after removing from -20°

Reaction Mix (Add in parenthetical order.)

1. 14.5μl H2O
2. 1μl plasmid DNA (1-2 μg) (1.419 μg/μl —> just use 1 μl)
3. 2μl Buffer IV (10x)
4. 2μl BSA (10x)
5. 10U/μl BsaI (0.5 μl for example)

——————————

20 μl final volume

(4) Gel- Plasmid Digestion

Gel Digestion

• 1.5% agarose gel
• 20 μl digestion vol + 3 μl loading dye
• run for 40 min @ 130 V
• take picture quickly! (avoid excessive UV exposure to nucleic acid)

(5) Gel- DNA Extration/Purification of Plasmid

DNA Extraction Kit (Gel Extraction) weigh eppendorf mct tube — slice out band from gel

• place in tube
• weigh again
• add volume of Buffer 1 equivalent to weight difference (gel slice weight) — incubate at 60°C for 5-10 min to let gel dissolve in extraction buffer

(should look yellow. pink/red means too basic)

• apply solution to column
• incubate for 1 min
• centrifuge @ 13,000 rpm, 1 min
• put column back to the original tube
• add 500 μl wash B (salt + EtOH)
• centrifuge 13,000 rpm 1-1.5 min
• put column back in column tube
• centrifuge @ 13,000 rpm, 2 min
• put column into new 1.5 mL Eppendorf microcentrifuge tube — add 20ul H2O to column
• incubate 1 min
• centrifuge @ 13,000 rpm, 1 min
• add 20ul H2O to column
• incubate 1 min
• centrifuge @ 13,000 rpm, 1min

(6) gRNA oligo ligation into plasmid

Reaction Mix

• 3μl sense/antisense annealed oligos
• 1μl purified vector (Kan. resistant)
• 1μl 10x Buffer (salts, ATP, DTT, etc.)
• 0.5μl ligase
• 4.5μl H2O

—————————— 

10μl final vol

Master Mixes (when doing many ligations simultaneously)

(1) Master Mix #1 (mix then pipette into PCR tubes)