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

1. Master Mixes (when doing many ligations simultaneously)

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

• 1 μl Vextor x (n+1)
• 0.8 μl 10x BF x (n+1)
• 3.2 μl H2O (n+1)

————————

• 5 μl / PCR tube final vol

1. 3 μl of oligo (pipette directly into PCR tubes with MM#1)

1. Master Mix #2 (mix then pipette into PCR tubes)

• 0.5μl enzyme x (n+1)
• 0.2μl 10x BF x (n+1)
• 1.3μl H2O x (n+1)

————————————

2 μl / PCR tube

4° C ON

(7) Transformation

• Use recombinant bacterial cells, strain DH5α (e. coli substrain) (in PCR tubes in -80°) - must ALWAYS keep on ice!!
• 4μl aliquot (ligation product) into common mix (-80C°) (~20-30 μl in common mix)
• incubate on ice for 25 min
• heat change —> 42°C in 1.5 min
• incubate on ice for 3.5 min
• put into Eppendorf w/ LB (no antibiotics - give cells a chance to express the plasmid gene against antibiotics)
• culture at 37°C for 1 hr
• centrifuge: 6,000 rpm / 1min
• trash most supernatant
• resuspend e. coli culture (w/ leftover LB) plate - LB + Kan antibiotics
• incubate 37° overnight

Note:

Kan 50 mg/L - high copy

Kan 15 mg/L - low copy

Notes:

1. If very efficient ligation / transformation with plasmid, then there will be an increased # of colonies that can survive. Therefore you should do a 2x plating (2x plates per transformation) so you can pick single colonies w/o contamination
2. ∴ 2x plates per PCR tube of transformed DH5α — 25 μl / plate —gradient plating
3. Akira prepares common stock of DH5α for the lab.
4. Use 4μl ligation product for newly ligated plasmids. If proven highly efficient, use 1μl of product.
5. 42°C incubation - use Jifeng’s ’42 Keep’ thermal cycler program
6. For Amp. resistance, LB culture is not necessary
7. For Amp res. plasmids, add 50-100μl of LB directly to PCR tube, then plate
8. After plating, allow to dry until no excess liquid
9. Culture in 37° incubator ON (16 hours max!!!) in semi open plastic bag to avoid excessive moisture loss

(8) Miniculture n (number of colonies you want to culture) —> n separate vials / per plate

Culture Solution Kan = 50 mg/mL (stock concentration)

—> 50 mg/L final dilution / concentration

ex. 100 mL PBS + 100 μl Kan

• use pipette tip to transfer colony gently to tube (can drop expel pipette tip directly into tube to be removed after centrifugation)
• 8 hours ON at 37°C

(9) Miniprep Protocol

• ON culture
• centrifuge 5000 rpm / 10 min discard supernatant
• add 250μl P1
• resuspend pellet in P1

• transfer to 2mL Eppendorf tubes
• add 250 μl P2
• mix by inverting until solution becomes blue

• add 350 μl N3 (guanidine hydrochloride acetyl acid)
• mix immediately by inverting tubes until solution (blue) turns colorless
• centrifuge 13,000 rpm / 10 min

• label QIAprep spin columns
• transfer supernatant to QIAprep spin columns
• incubate / let stand for 1 min
• centrifuge @ 13,000 rpm / 2 min
• discard spin-through
• + 750 μl PE to column
• centrifuge @ 13,000 rpm / 2 min
• + 750 μl PE to column
• centrifuge @ 13,000 rpm / 2 min
• vacuum columns’ inner rim/ridge (do NOT vacuum membrane) remove columns
• label 1.5mL Eppendorf tubes
• places columns into 1.5mL Eppendorf tubes
• add 50 μl dH2O
• let stand 1 min
• centrifuge 1 min
• store samples at -20°C

(10) Miniprep Sample Sequencing

Sequencing reaction

• 2 Kb plasmid

requires ~100 ng [ ] per tube

• _____ ng/μl [ ]

ex. 170 ng/μl [ ]

0.7μl sample

4.3μl H2O

• for multiple samples around 170ng/μl [ ], use 4.3μl H2O and then add appropriate concentration of sample to sequencing PCR tube
• M13_F primer
• max read length
• check sequences for gRNA inserts

(11) Plasmid gRNA PCR Amplification

Mix 0.2μl sample

0.3μl For

0.3μl Rev

25μl Phusion MasterMix (with NTs and Taq) *** 24.2μl ddH2O

—————————

50 μl total vol

• • • 2x Phusion 9c ‘MasterMix’ (Thermo) #F-532

Master Mix

• (n + 0.5) x each component
• use pipette tips with filters
• For + Rev + Phusion MM + ddH2O
• label sides + top of tubes (#1 - #n)
• briefly centrifuge samples + primers + Phusion (once dissolved)
• add For + Rev + Phusion MM + ddH2O into ‘MM’ tube
• pipette up and down to mix (~5x times)
• aliquot 49.8 μl of MM into each PCR tube
• add 0.2 μl sample to each tube
• put in / start thermalcycler

(12) QIAquick PCR Purification Kit

• 5 volumes Buffer PB to 1 vol PCR rxn (add directly to PCR tube)
• mix

(if orange or violet, add 10μl 3M sodium acetate and mix)

(mix should turn yellow)

• place QIAquick columns in 2mL collection tubes

• bindDNA - add sample to column
• incubate for 1 min
• centrifuge for 30-60 sec
• discard flow through + put column back in 2mL tube

• wash - add 0.75mL PE to column
• centrifuge for 30-60 sec
• discard flow-through + put column back in 2 mL tube
• centrifuge QIAquick column for 1 min in 2 mL collection tube
• vacuum rim of column (10μl tip + 1mL tip + vac system)
• centrifuge QIAquick column for 1 min in 2 mL collection tube

• place columns in RNase free 1.5mL microcentrifuge tubes (put lids on column tubes and cut the column tubes’ lids off)
• eluteDNA - add 20μl RNase free H2O to centre of column centrifuge column for 1 min
• measure concentration (nanodrop)

(13) Purified PCR product gel 1μl purified product

1μl dye

5/6 μl TBE (1x)

———————————— 

8/9 μl total vol

(2μl Red / 50 mL of gel solution)

(14) MEGAscript Kit (amnion / life technologies) 

Prep

• prepare 70% EtOH - minimum 3mL / sample (for day 2)
• keep RNAPol on ice at all times
• thaw frozen reagents
• vortex 10x RBuffer and NTPs
• briefly centrifuge
• (NTPs should be kept on ice. 10x RB must be kept at room temp.)

Assemble Txn Rxn

Sample

Use ~500ngof Miniprep sample —> use Nanodrop concentration to calculate volume

MasterMix

Add MM components in the following order: H2O, NTPs, 10x BF, enzyme mix, MPI T7

(n = number of samples + 0.5)

n x 2μl ATP

n x 2μl CTP

n x 2μl GTP

n x 2μl UTP

n x 2μl 10x Buffer

n x ___μl H2O

n x 0.5μl MPIT7 (separate - Max Planck purified T7 enzyme)

n x 2μl Enzyme Mix

___μl MasterMix + ___μl sample = 20μl

• add sample volume directly to PCR tubes
• add MasterMix to samples
• mix thoroughly
• incubate in thermalcycler, 37°C, overnight (max 16 hrs.)

Day 2

TURBO DNase

• add 1μl TURBO DNase
• mix well
• incubate further 15 min at 37°C
• label RNase free 1.5mL microcentrifuge (μc)tubes

RNA Recovery - LiCl Precipitation

precipitate RNA

• add 30μl RNase free H2O
• add 30μl LiCl precipitation solution

(preferred method: add LiCl to newly labeled 1.5mL μc tubes, add 30μl H2O to samples, transfer H2O + samples (50μl) to the μC tubes with LiCl) *mix thoroughly

• chill >30 min at -20°C
• centrifuge: 4°C, max speed, 15 min
• CAREFULLY remove supernatant
• wash pellet with 1mL 70% EtOH
• centrifuge: 4°C, max speed, 15 min
• wash pellet with 1mL 70% EtOH
• centrifuge: 4°C, max speed, 15 min
• wash pellet with 750μl 70% EtOH (lower vol. allows for easier removal of final wash) (3x total washes - centrifuge between each wash)
• centrifuge: 4°C, max speed, 15 min
• carefully remove EtOH
• air dry 5-10 min
• resuspend RNA in~50μl RNase free H2O (Variable volume. If some pellets are smaller, use less H2O.) (make very sure pellet is fully resuspended - tricky because appears clear)
• determine concentration using Nanodrop (remember to use RNA detection)
• store at -80°C

(15) gRNA - Cas9 complex formation

Injection / electroporation complex

10-20μg protein (from prep 5 μg/μl)

5μg each gRNA (concentration dependent)

0.9 μg 10x Cas9 Buffer

__ μl H2O

———————————

10 μl final volume

• Microinjection (single cell stage complex)

5μg protein (around 1 μl - from prep 5μg/μl)

4 μg gRNA ([] dependent)

0.9μl 10x Cas9 Buffer

__μl Hzo

1 μl Fastgreen

———————————

10μl final volume

• Add in seq.

1. H2O
2. Buffer
3. gRNA
4. Protein

• aliquot 5 ———————————l into 2x 1.5 mL PCR tubes (hyper sterile)
• incubate at room temp for 5 min.
• store at -80°C