Feb 14, 2022

Public workspaceDetermining Genome Targeting Efficiency using T7 Endonuclease I (M0302) V.4

DOI
dx.doi.org/10.17504/protocols.io.bfhqjj5w
  • 1New England Biolabs
  • New England Biolabs (NEB)
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DOI: dx.doi.org/10.17504/protocols.io.bfhqjj5w
External link: https://www.neb.com/protocols/2014/08/11/determining-genome-targeting-efficiency-using-t7-endonuclease-i
Protocol Citation: New England Biolabs 2022. Determining Genome Targeting Efficiency using T7 Endonuclease I (M0302). protocols.io https://dx.doi.org/10.17504/protocols.io.bfhqjj5wVersion created by New England Biolabs
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: April 23, 2020
Last Modified: February 14, 2022
Protocol Integer ID: 36112
Keywords: T7 Endonuclease I, targeting efficiency,
Abstract
T7 Endonuclease I recognizes and cleaves non-perfectly matched DNA. This protocol describes how to determine genome targeting efficiency by digesting annealed PCR products with T7 Endonuclease I. In the first step PCR products are produced from the genomic DNA of cells whose genomes were targeted using Cas9, TALEN, ZFN etc. In the second step, the PCR products are annealed and digested with T7 Endonuclease I. Fragments are analyzed to determine the efficiency of genome targeting.
Guidelines
REFERENCES:
CITATION
Guschin DY, Waite AJ, Katibah GE, Miller JC, Holmes MC, Rebar EJ (2009). A rapid and general assay for monitoring endogenous gene modification.. Methods in molecular biology (Clifton, N.J.).
LINK
https://doi.org/10.1007/978-1-60761-753-2_15

Materials
MATERIALS
ReagentQ5 Hot Start High-Fidelity 2X Master Mix - 100 rxnsNew England BiolabsCatalog #M0494S
ReagentT7 Endonuclease I - 250 unitsNew England BiolabsCatalog #M0302S
Required Materials:
  • Q5® Hot Start High-Fidelity 2X Master Mix (M0494S)
  • T7 Endonuclease I (M0302S)
  • 0.25 mM EDTA
  • Purified genomic DNA from targeted cells
  • PCR primers to amplify a ~1kb region containing the target site
  • The target site should be offset from the center of the amplicon so that digestion produces easily resolvable DNA fragments
  • PCR primer design is critical. Please visit NEB's Tools and Resources page to optimize your primer design using the NEB Tm Calculator
  • A PCR thermocycler with programmable temperature ramp rate
  • DNA purification system - we recommend Ampure XP beads
  • Apparatus to quantitate DNA - spectrophotometer or fluorometer
  • Apparatus to analyze DNA fragments – e.g. Agilent Bioanalyzer, Qiagen Qiaxel, or standard agarose gel electrophoresis
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
PCR
PCR
Set up a Amount50 µL PCR reaction using ~Amount100 ng genomic DNA as a template. For each amplicon set up 3 PCR reactions using the following templates:
  • gDNA from targeted cells (e.g. Cas9, or TALEN transfected cells)
  • gDNA from negative control cells (e.g. non-specific DNA transfected cells)
  • water (i.e. no template control)
PCR using Q5 High-Fidelity DNA Polymerase
ABC
COMPONENT50 µl REACTIONFINAL CONCENTRATION
Q5® Hot Start High-Fidelity 2X Master Mix (M0494)25 µl1X
10 µM Forward Primer2.5 µl0.5 µM
10 µM Reverse Primer2.5 µl0.5 µM
Template DNAvariable100 ng total
Nuclease-free waterTo 50 µl

Pipetting
Gently mix the reaction.
Mix
Collect all liquid to the bottom of the tube by a quick spin if necessary.
Transfer PCR tubes to a PCR machine and begin thermocycling:
Cycling Conditions
ABC
STEPTEMPERATURETIME
Initial Denaturation98°C30 seconds
35 cycles98°C5 seconds
*50-72°C10 seconds
72°C20 seconds
Final Extension72°C2 minutes
Hold4-10°C
*Use of the NEB Tm Calculator is highly recommended.

Note
Note: Q5 Hot Start High-Fidelity 2X Master Mix does not require a separate activation step. Standard Q5 cycling conditions are recommended.
PCR
Analyze a small amount of the of the PCR product to verify size and appropriate amplification.
Analyze
Purify the PCR reaction using Amount90 µL Ampure XP beads following the manufacturer’s recommendations.
Note
Other PCR purification systems (e.g. Monarch PCR & DNA Clean Up Kit, or Zymo DNA Clean and Concentrator™) are acceptable.

Elute PCR products in Amount30 µL water , recovering Amount25 µL .
Measure the concentration of the purified PCR products.
Analyze
T7 Endonuclease I digestion
T7 Endonuclease I digestion
Assemble reactions as follows:
AB
COMPONENT19 µl ANNEALING REACTION
DNA200 ng
10X NEBuffer 22 µl
Nuclease-free WaterTo 19 µl

Pipetting
Anneal the PCR products in a thermocycler using the following conditions:
Hybridization Conditions
ABCD
STEPTEMPERATURERAMP RATETIME
Initial Denaturation95°C5 minutes
Annealing95-85°C-2°C/second
85-25°C-0.1°C/second
Hold4°CHold

Incubation
Add Amount1 µL T7 Endonuclease I (M0302) to the annealed PCR products for a final volume of Amount20 µL .
Pipetting
Incubate at Temperature37 °C for Duration00:15:00 .
Incubation
Stop the reaction by adding Amount1.5 µL 0.25 M EDTA .
Pipetting
Purify the reaction using Amount36 µL Ampure XP beads according to the manufacturer’s suggestion.
Note
This step is optional since 1 µl of the reaction will not interfere with analysis on an Agilent Bioanalyzer using DNA1000 reagents.

Optional
Elute the DNA fragments in Amount20 µL water , recovering Amount15 µL .
Analysis
Analysis
Analyze the fragmented PCR products and determine the percent of nuclease-specific cleavage products (fraction cleaved).
Analyze
Calculate the estimated gene modification using the following formula:
% gene modification = 100 x (1 – (1- fraction cleaved)1/2)
Computational step
Citations
Guschin DY, Waite AJ, Katibah GE, Miller JC, Holmes MC, Rebar EJ. A rapid and general assay for monitoring endogenous gene modification.
https://doi.org/10.1007/978-1-60761-753-2_15