Determining Genome Targeting Efficiency using T7 Endonuclease I (M0302)
Set up a 50 µl PCR reaction using ~100 ng of genomic DNA as a template.
For each amplicon set up 3 PCR reactions (positive, negative, no-template control). 
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.
Analyze a small amount of the of the PCR product to verify size and appropriate amplification.
Purify the PCR reaction using 90 µl of Ampure XP beads following the manufacturer’s recommendations.
Elute PCR products in 30 µl of water, recovering 25 µl.
Measure the concentration of the purified PCR products.
Assemble reactions as follows: Anneal the PCR products in a thermocycler (see guidelines for conditions). 
Add 1 µl of the T7 Endonuclease I to the annealed PCR products. 
Incubate at 37°C for 15 minutes. 
Stop the reaction by adding 1.5 µl of 0.25 M EDTA.
Purify the reaction using 36 µl of Ampure XP beads according to the manufacturer’s suggestion.
Elute the DNA fragments in 20 µl of water, recovering 15 µl.
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)
