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===Dpn II & USER enzyme digestion=== reaction system 1: *To 50ul of ssDNA, add 1.5ul USER enzyme, 37C 1hr. *Add 6ul 10X DpnII buffer, 2ul 100uM DpnII-V4 guide oligo *94C5min -> 60C 5min -> turn off and wait for 20min -> add 1ul Dpn II (50K/ml)-> 37C 1h. *Add 1ul USER enzyme -> 37C 1h -> 75C 20min *PAGE purification in 6% TB-Urea 2D gels. *DNA were eluted from the shearing gel assembly and Nanosep columns. *Ethanol purification and PAGE gel quantification. reaction system 3&4: *To 80ul of ssDNA, add 4ul USER enzyme, 37C 1hr. *Add 10ul 10X DpnII buffer, 2ul 100uM DpnII-V4 guide oligo *94C5min -> 60C 5min -> turn off and wait for 20min -> add 2ul Dpn II (50K/ml) -> 37C 1h. *Add 2ul USER enzyme -> 37C 1h -> 75C 20min *PAGE purification in 6% TB-Urea 2D gels. *DNA were eluted from the shearing gel assembly and Nanosep columns. *Ethanol purification and PAGE gel quantification. yield: 5ng/ul *80ul I ran a gel to quantify the gel along with the PCR product. Then I found that batch template 2 is not working. Therefore half of the plate is actually not working, thus final percent yield is too low.
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