PROTOCOL

Edit-R™ lentiviral sgRNA
positive controls

Product description
The following protocol is an example of thermal cycling and cleavage
conditions for a DNA mismatch detection assay using T7 Endonuclease I(T7EI) and the Edit-R Lentiviral sgRNA Control primer set utilizing genomic
DNA (gDNA) from direct cell lysis. For direct cell lysis, cells were treated forgene editing in a 96-well format.

Phusion Hot Start II High-Fidelity
DNA Polymerase (2 U/µL)

1 µL

0.04 U/µL

Water, nuclease free32 µL

N/A

Cell lysate

5 µL

N/A4. Thermal cycling condition
1. Lyse cells in 100 µL of 1x Phusion High-Fidelity buffer with additives

Cycle step

Temperature

TimeCycle(s)Initial denaturation

98 °C

3 min

1Denature

98 °C

10 s

Touchdown annealing

72 °C–1 °C/cycle

15 s

0.5 mg/mL

Extension

72 °C

30 s

N/A

Denature

98 °C

10 s

Annealing

62 °C

15 s

Extension

72 °C

30 s

Final extension

72 °C

10 minReagent

VolumeFinal concentration

5x Phusion HF Buffer

20 µL

1xProteinase K (~ 20 mg/mL)

5 µL~ 1 mg/mL

RNase A (10 mg/mL)

5 µL

Water, nuclease free70 µL

2. Seal the 96-well plate with a plate seal to minimize evaporation andcross contamination. Incubate for 15–30 minutes at 56 °C, followed bydeactivation for 5 minutes at 96 °C. Briefly centrifuge plate to collect liquid
at bottom of wells.
3. Set up 50 µL PCR for each sample to be analyzed.
Note: Other PCR reagents can be used provided that PCR
optimization is performed for mismatch detection conditions andtesting compatibility with direct cell lysis or purified gDNA.
Reagent

VolumeFinal concentration

5x Phusion HF buffer

10 µL

1x

Forward primer (50 µM)

0.5 µL500 nM

Reverse primer (50 µM)

0.5 µL500 nMdNTPs (10 mM)

1 µL

200 µM each1025
1

5. Heat PCR samples to 95 °C for 10 minutes and then slowly (> 15 minutes)
cool to room temperature (~ 25 °C).
6. PCR product (~ 5 µL) can be run on 2% agarose gel to confirm
amplification.
7. Set up 15 µL reactions for mismatch detection assay using T7EI. Be sureto include at least one sample without the T7EI enzyme (No enzymenegative control) and/or Untreated (no gene editing negative control).
Note: Other mismatch detection assays can be used following themanufacturer’s recommended protocol

dharmacon.horizondiscovery.com

Reagent

VolumeFinal concentration

Materials

PCR from gDNA (300-500 ng/reaction)

10 μL

variable

Water, nuclease free

3 μL

N/A

NEBuffer 2 (10x)1.5 μL

1xT7 Endonuclease I (10 U/µL)

0.5 μL

0.33 U/μL• Thermo Scientific Phusion Hot Start II High-Fidelity DNA Polymerase, 2U/μL (Cat #F-549S)• T7 Endonuclease I, 10 U/µL (NEB, Cat #M0302S)• Thermo Scientific Proteinase K,~ 20 mg/mL (Cat #EO0492)• Thermo Scientific RNase A, 10 mg/mL (Cat #EN0531)• Thermo Scientific 5x Phusion HF Buffer (Cat #F-518L)• NEBuffer 2 (NEB, Cat #B7002S)

8. Incubate for 25 minutes at 37 °C.
9. Immediately run entire reaction volume with appropriate gel loading
buffer on 2% agarose gel, since T7EI enzyme cannot be heat inactivated.
Compare samples with expected gene editing to the No enzyme and/orUntreated negative controls.

Expected band sizes (bp)Genetarget

Species Primer

UK-007050-01-xx

PPIB

Human

UK-007150-01-xx

Ppib

UK-007250-01-xx

Ppib

UK-007060-01-xx

DNMT3B

UK-007160-01-xx

Dnmt3b

UK-007260-01-xx

Dnmt3bSpeciesSequenceTm* (°C) MW

Extinction coefficient
No editing

Forward
ReverseForward
Mouse
ReverseForwardRat
ReverseForward
Human
ReverseForward
Mouse
ReverseForwardRat
ReverseGAACTTAGGCTCCGCTCCTT
CTCTGCAGGTCAGTTTGCTG
CCAGCCTGTTTGTTGGATTT
ACTGTGAGGCTGCCAGATTTTGCCATTTCCTCACTCCCTA
GGCTACCGCTACAAATGGACTGAGAAGGAGCCACTTGCTTGACCAAGAACGGGAAAGTCA
ACTTGGTGATTGGTGGAAGCTTAAGCCACACCCTCCTCTG
GGCATTGATCCCATGTAACC
CTCCGAGCTGCTAAGAGGAA

64.0
64.3
64.2
64.2
60.4
62.4
64.4
64.4
64.3
64.5
60.4
62.4

6044.06115.06105.16148.15938.96111.06157.16193.16228.15972.96077.06151.0176184
178151176905191297165727
197820197197220800199164174218189330201754

505
572
524
544
561
532

With editing~ 330,~ 174~ 409,~ 163~ 342,~ 170~ 335,~ 209~ 372,~ 189~ 319~ 212*Tm calculated specifically for use with Phusion DNA polymerases. Tm should be calculated for specific DNA polymerases and PCR reagents using appropriate parameters.

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
Horizon Discovery, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Phusion Hot Start II High-Fidelity, Proteinase K, RNase A, and 5x Phusion HF Buffer is a registered trademark of Thermo Scientific, Inc.
T7 Endonuclease I and NEBuffer 2 is a registered trademark of NEB. All trademarks are the property of Horizon Discovery Company unlessotherwise specified. ©2018 Horizon Discovery Group Company—All rights reserved. First published March 2015. UK Registered Head Office:
Building 8100, Cambridge Research Park, Cambridge, CB25 9TL, United Kingdom.

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