GRNA Production

From Tanaka Wiki
Jump to: navigation, search

(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)