Determining Genome Targeting Efficiency using T7 Endonuclease I (M0302)
New England Biolabs
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.
Before start
Steps
PCR
Set up a 50µL PCR reaction using ~100ng 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
| A | B | C |
|---|---|---|
| COMPONENT | 50 µl REACTION | FINAL CONCENTRATION |
| Q5® Hot Start High-Fidelity 2X Master Mix (M0494) | 25 µl | 1X |
| 10 µM Forward Primer | 2.5 µl | 0.5 µM |
| 10 µM Reverse Primer | 2.5 µl | 0.5 µM |
| Template DNA | variable | 100 ng total |
| Nuclease-free water | To 50 µl |
Gently mix the reaction.
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
| A | B | C |
|---|---|---|
| STEP | TEMPERATURE | TIME |
| Initial Denaturation | 98°C | 30 seconds |
| 35 cycles | 98°C | 5 seconds |
| *50-72°C | 10 seconds | |
| 72°C | 20 seconds | |
| Final Extension | 72°C | 2 minutes |
| Hold | 4-10°C |
*Use of the NEB Tm Calculator is highly recommended.
Analyze a small amount of the of the PCR product to verify size and appropriate amplification.
Purify the PCR reaction using 90µL following the manufacturer’s recommendations.
Elute PCR products in 30µL, recovering 25µL.
Measure the concentration of the purified PCR products.
T7 Endonuclease I digestion
Assemble reactions as follows:
| A | B |
|---|---|
| COMPONENT | 19 µl ANNEALING REACTION |
| DNA | 200 ng |
| 10X NEBuffer 2 | 2 µl |
| Nuclease-free Water | To 19 µl |
Anneal the PCR products in a thermocycler using the following conditions:
Hybridization Conditions
| A | B | C | D |
|---|---|---|---|
| STEP | TEMPERATURE | RAMP RATE | TIME |
| Initial Denaturation | 95°C | 5 minutes | |
| Annealing | 95-85°C | -2°C/second | |
| 85-25°C | -0.1°C/second | ||
| Hold | 4°C | Hold |
Add 1 to the annealed PCR products for a final volume of 20µL.
Incubate at 37°C for 0h 15m 0s.
Stop the reaction by adding 1.5µL.
Purify the reaction using 36µL according to the manufacturer’s suggestion.
Elute the DNA fragments in 20µL, recovering . 15µL.
Analysis
Analyze the fragmented PCR products and determine the percent of nuclease-specific cleavage products (fraction cleaved).
Calculate the estimated gene modification using the following formula:
% gene modification = 100 x (1 – (1- fraction cleaved)1/2)
