Protocol

Dharmacon™ E-dit-R™ guideRNA control kits via T7EI assay

Version: 1May 2024

For research only. Not for use in diagnostic procedures.

Product Description
The Edit-R control kits provide validated guide RNAs and primer pairs for detection of cleavage for the designatedhousekeeping gene or safe harbor region.
These gene-specific positive control kits and region-specific cutting control kits are validated for mismatch detection
assays to verify gene editing experiments. 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 control primer set utilizing genomic
DNA (gDNA) from direct cell lysis. The same primer pairs can also be used for “Tracking of Indels by Decomposition” (TIDE)
analysis. The volumes in this protocol are for direct cell lysis in a 96-well plate format.

T7EI Protocol
1.

Lyse cells in 100 µl of 1x Phusion High-Fidelity buffer with additives.
Reagent

VolumeFinal concentration

5x Phusion HF Buffer

20 µL

1xProteinase K (~ 20mg/mL)

5 µL~ 1 mg/mL

RNase A (10 mg/mL)

5 µL

0.5 mg/mL

Water, nuclease free70 µL

N/A

2.

Seal the 96-well tissue culture plate with a plate seal to minimize evaporation and cross contamination. Incubate for
15-30 minutes at 56 °C.

3.

Transfer cell lysate into a 96-well PCR-plate, seal plate with a plate seal, and place into a thermocycler for 5 minutes at
96 °C. Briefly centrifuge the plate to collect liquid at the bottom of each well.
Note: Due to variable depth of thermocycler heat blocks, deactivation time may need to be increased to 15 or 30minutes.
4.

Set up 50 µl PCR reaction for each sample to be analyzed.

Note: Other PCR reagents can be used provided that PCR optimization is performed for mismatch detection

conditions and testing compatibility with direct cell lysis or purified gDNA.

1Reagent

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

1 µL

0.04 U/µL

Water, nuclease free

32 µL

N/A

Cell lysate

5 µL

N/A5.

Thermal cycling conditions:
Cycle step

Temperature

Time Cycles

Initial denaturation

98 °C

3 min

Denature

98 °C

10 s

Touchdown annealing

72 °C – 1 °C/cycle

15 s

Extension

72 °C

30 s

Denature

98 °C

10 s

Annealing

62 °C

15 s

Extension

72 °C

30 s

Final extension

72 °C

10 min110251

6.

Heat PCR samples to 95 °C for 10 minutes and then slowly (>15 min) cool to room temperature (~25 °C).

7.

PCR product (~5 µl) can be run on a 2% agarose gel to confirm amplification.

8.

Set up 15 µl reactions for mismatch detection assay using T7EI. Be sure to include at least one sample without theT7EI enzyme (No enzyme negative control and/or untreated (no gene editing negative control).
Note: Other mismatch detection assays can be used following the manufacturer’s recommended protocol.

9.

Incubate for 25 minutes at 37 °C.

10. Immediately run entire reaction volume with appropriate gel loading buffer on 2% agarose gel, since the T7EI enzyme
cannot be heat inactivated. Compare samples with expected gene editing to the no enzyme and/or untreatednegative controls.

Materials•

Thermo Scientific Phusion Hot Start II High-Fidelity DNA Polymerase, 2 U/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)

2Reagent

Volume Finalconcentration

PCR from gDNA (300-500ng/reaction)

10 μLvariable

Water, nuclease free

3 μL

N/A

NEBuffer 2 (10x)1.5 μL

1x

T7 Endonuclease I (10 U/µL)

0.5 μL

0.33 U/μL

Expected band sizes (bp)Target gene/region

PPIB

DNMT3BAASVI_sg1

AASV1_sg2

AASV1_sg3

Ppib

Species

PrimerPrimer sequence

Tm*(°C)

Human GUGUAUUUUGACCUACGAAU Forward
ReverseGAACTTAGGCTCCGCTCCTT

64.0

CTCTGCAGGTCAGTTTGCTG

64.3

HumanForward
ReverseTGAGAAGGAGCCACTTGCTT

64.4

GACCAAGAACGGGAAAGTCA

64.4

Forward
ReverseACTAGCTGAGCTCTCGGACC

63.1GCTGCCCAAGGATGCTCTTT

68.0

Forward
ReverseGGCCAGGCAGATAGACCAGA66.9

CCAACGCCGACGGTATCAG

69.1

Forward
ReverseGAACCCAGCGAGTGAAGACG

67.6

CTCTTCCGATGTTGAGCCCC

68.4

Forward
Reverse

CCAGCCTGTTTGTTGGATTT

64.2

ACTGTGAGGCTGCCAGATTT

64.2

Forward
ReverseACTTGGTGATTGGTGGAAGC

64.3

TTAAGCCACACCCTCCTCTG

64.5

Human

Human

Human

Mouse

Dnmt3bGuide RNA target sequence

Mouse

GCUGAAUUACUCACGCCCCAGACGCAAGGGAGACATCCGTGCGGCTCCAATTCGGAAGTG

GAGTTCCGGCGGCACAGCAAGUAUACUUUGAUUUACAAAUGCUGAAUUAUACCCGCCCCA

NoeditingWith editing505~ 330~ 174544~ 335~ 209

481~272~209514~303~211590~427~163572~409~163561~372~189*Tm calculated specifically for use with Phusion DNA polymerases. Tm should be calculated for specific DNA polymerases andPCR reagents using appropriate parameters.

For more information
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DHARMACON is a trademark of Dharmacon Inc.

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