Chemidoc xrs device

The primers listed in the Table 2 were used for PCR amplification to test the presence of gRNA inserts on plasmids. The primer for Cloning Forward was common for both gRNA inserted plasmids, annealing U6 promoter upstream of the gRNA cloning site. For each gRNA, original reverse oligos used annealing of gRNA were used as reverse primers for PCR reaction. Stock primers (100 µM) were diluted to 1:20 using nuclease free water. PCR reaction was set according to the instructions. 10 µl of 2X Master Mix, 1 µl of Plasmid DNA, 1 µl of Forward Primer and Reverse Primer, 7 µl of Nuclease Free Water. Thermocycler was adjusted as Initial denaturation at 95°C for 60 sec, 25 cycles of (Denaturation at 95°C for 15 sec 25 cycles, and Annealing at 55°C for 15 sec, Extension at 68°C for 10 sec), and final extension at 68°C for 2 minutes. 2% agarose gel was prepared using TBE buffer. Into each PCR product, 4 µl of 6X loading dye was added. 4 µl of 50 bp DNA Step Ladder was loaded into the first well. PCR products were loaded as 10 µl in each well. Gel was run at 150 V for 35 minutes. Gel image was obtained from Biorad Chemidoc XRS device. Plasmids isolated by midiprep protocol were sent to Medsantek, Turkey for sequencing. Directional Sanger sequencing was performed using 10 µM primer Cloning Forward given in Table 2 was used. The plasmid DNA concentrations were adjusted to 100 ng/µl.

Thermocycler was used to denature and anneal 5 µl of PCR product as recommended by manufacturer (EnGen™ Mutation Detection Kit, NEB, E3321S). Thermocycler was adjusted for heteroduplex formation as following:
Denaturation at 95°C for 5 minutes, annealing at 95-85°C at 1.5°C/min ramp rate for 5 sec, annealing at 85-25°C at 0.1°C/min ramp rate for 5 sec and Hold at 4°C until use. To digest the heteroduplex, 1 µl T7 Endonuclease I was added into the mixture and incubated for 20 minutes at 37°C in the thermocycler. Afterwards, 1 µl of Proteinase K was added to inhibit T7E activity and incubated 37°C for 5 minutes in the thermocycler. Sample was mixed with 4 µl of Purple Gel Loading dye (6X) and loaded on 1% agarose gel prepared with TBE Buffer and run at 150 V for 45 minutes. Gel image was obtained from Biorad Chemidoc XRS device. ImageJ was used to quantify each product with band density function as we have done previously (Mahmoud et al., 2013) to determine % of indels. To this end, digested band densities and non-digested band intensities quantified. Digested band density divided by total band density to determine the percentage of indel formation.