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 )
