PROTOCOL

Dharmacon™ Edit-R™ Cas9 mRNAand synthetic guide RNAtransfection protocol

The following is an abbreviated protocol for transfecting Dharmacon™
Edit-R™ Cas9 mRNA (Cat #CAS11195, #CAS11859, or #CAS11860) withsynthetic guide RNA into cultured mammalian cells using DharmaFECT™
Duo transfection reagent (Cat #T-2010-xx). Synthetic guide RNA can beeither Edit-R synthetic tracrRNA (Cat #U-002005-xx) complexed with crRNA(predesigned or custom) or Edit-R synthetic single guide RNA (sgRNA,custom). Intended for use after optimization for your cell line has been
completed. For full details, as well as optimization guidelines please see theTechnical Manual.
Tube 1

Tube 2

DiluteDharmaFECT Duotransfection reagent
in serum-freemedium

crRNA:tracrRNA orsynthetic sgRNA +Cas9 mRNA

10 µL

10 µL

Incubate at RT for 5 min

Combine Tubes 1 and 2
Carefully mix by pipetting

20 µL

Incubate at RT for 20 min
Add 80 µL growth medium

96–well protocol
Day 1
Cell plating

Seed cells at a density that gives 70-90% confluency on the next dayDay 2Prepareworking
solutions ofreagents fortransfectionCombineworking
solutions fortransfection
mixturesynthetic guide
RNADilute and mix crRNA and tracrRNA to a working
concentration of 2 μM in 10 mM Tris-HCl pH 7.4
or Dilute synthetic sgRNA to a working
concentration of 2 µM in 10 mM Tris-HCl pH 7.4

Cas9 mRNA

Dilute Cas9 mRNA to a working concentrationof 100 ng/µL in serum-free medium
For one well

For mulitple wells

synthetic guideRNA1.25 μL_ μL

Cas9 mRNA2 μL_ μL

Serum-freemedium

To 10 μL

_ μL

DharmaFECT Duotransfection
reagent

0.1–0.8 μL_ μL

Serum-freemedium

To 10 μL

_ μL

Tube 1Tube 2Prepare
working
solution ofDharmaFECTDuo fortransfection

Incubate at room temperature for 5 minutes before next step
Combine Tube 1 and Tube 2 and carefully mix by pipeting

100 µL

Replace growth medium oncells with transfection mixture

Combinetransfection
mixtureTransfectcells

dharmacon.horizondiscovery.com

Incubate at room temperature for 20 minutes before next step
Add full growth
medium

80 μL

_ μL

Total

100 μL_ μL

Replace growth medium on cells with 100 μL of transfection mixture

24–well protocol6–well protocol

Day 1

Day 1

Cell plating

Seed cells at a density that gives 70-90% confluency on the next day

Day 2
Prepareworkingconcentration
solutions ofmaterials fortransfection

Prepareworkingconcentration
DharmaFECTDuo fortransfection

synthetic guide RNA

Dilute and mix crRNA and tracrRNA to aworking concentration of 2.5 μM in 10 mM
Tris-HCl (pH7.4) or Dilute synthetic sgRNA toa working concentration of 2 µM in 10 mM
Tris-HCl pH 7.4

Cas9 mRNA

Dilute Edit-R CRISPR-Cas9 mRNA to aworking concentration of 100 ng/µL in
serum-free medium
For mulitple wells

Tube 1synthetic guide RNA

5 μL

_ μL

Cas9 mRNA

10 μL

_ μL

Serum-free medium

To 50 μL

_ μL

Tube 2DharmaFECT Duotransfection reagent

0.5–0.8 μL_ μL

Serum-free medium

To 50 μL

_ μL

Incubate at room temperature for 5 minutes before next step

Prepareworking
concentration
solutions ofmaterials fortransfection

Combineworking
concentration
solutions fortransfection
mixturePrepareworking
concentration
DharmaFECTDuo fortransfection

Transfect cells

Incubate at room temperature for 20 minutes before next step
Add full growth
medium

400 μL_ μL

Total

500 μL_ μL

Replace growth medium on cells with 100 μL of transfection mixture

synthetic guide RNA

Dilute and mix crRNA and tracrRNA to aworking concentration of 2.5 μM in 10 mM
Tris-HCl (pH7.4) or Dilute synthetic sgRNA
to a working concentration of 2 µM in
10 mM Tris-HCl pH 7.4

Cas9 mRNA

Dilute Edit-R CRISPR-Cas9 mRNA to aworking concentration of 100 ng/µL inserum-free medium
For one well

For mulitple wells

synthetic guide RNA25 μL_ μLCas9 mRNA50 μL_ μL

Serum-free medium

To 250 μL_ μL

DharmaFECT Duotransfection reagent

2.5–20 μL_ μL

Serum-free medium

To 250 μL_ μL

Tube 1Tube 2Incubate at room temperature for 5 minutes before next step
Combine Tube 1 and Tube 2 and carefully mix by pipeting

Combine Tube 1 and Tube 2 and carefully mix by pipeting
Preparetransfection
mixtureSeed cells at a density that gives 70-90% confluency on the next day

Day 2

For one well
Combineworking
concentration
solutions fortransfection
mixtureCell plating

Preparetransfection
mixtureTransfect cells

Incubate at room temperature for 20 minutes before next step
Add full growth
medium

2,000 μL

_ μL

Total

2,500 μL

_ μL

Replace growth medium on cells with 100 μL of transfection mixture

t +44 (0) 1223 976 000 (UK) or +1 800 235 9880 (USA); +1 303 604 9499 (USA)f + 44 (0) 1223 655 581
w horizondiscovery.com/contact-us or dharmacon.horizondiscovery.com/service-and-support
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All trademarks are the property of Horizon Discovery Company unless otherwise specified. ©2018 Horizon Discovery Group Company—All rightsreserved. First published October 2017. UK Registered Head Office: Building 8100, Cambridge Research Park, Cambridge, CB25 9TL, United Kingdom.

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