Gel doc gel imaging system

Purified ADV PCR products (400 ng) amplified from the genomic DNA extraction were re-annealed and treated with SURVEYOR nuclease (Transgenomics, Omaha, NE, USA) according to the manufacturer's recommended protocol. The products were analyzed on 10% TBE polyacrylamide gels, which were stained with SYBR Gold DNA stain (Life Technologies) and imaged using a Bio-Rad Gel Doc gel imaging system (Richmond, CA, USA). Quantification was based on the relative band intensities, as described by Cong et al. (2013) [12] (link). The PCR products were ligated into the pMD20-T vector (Takara Bio Inc.) and submitted for sequencing using universal primers (BGI, Guangzhou, China).

Genomic DNA was extracted using the QuickExtract DNA Extraction Solution (Epicenter) following the manufacturer’s protocol. The genomic region flanking the CRISPR target site for each gene was PCR amplified, and products were purified using QiaQuick Spin Column (QIAGEN) following the manufacturer’s protocol. 200–500 ng total of the purified PCR products were mixed with 1 μl 10 × Taq DNA Polymerase PCR buffer (Enzymatics) and ultrapure water to a final volume of 10 μl and were subjected to a re-annealing process to enable heteroduplex formation: 95 °C for 10 min, 95–85 °C ramping at −2 °C/s, 85–25 °C at −0.25 °C/s, and 25 °C hold for 1 min. After re-annealing, products were treated with T7 Endonuclease I (NEB) following the manufacturer’s recommended protocol and analyzed on 4–20% Novex TBE polyacrylamide gels (Life Technologies). Gels were stained with SYBR Gold DNA stain (Life Technologies) for 10 min and imaged with a Gel Doc gel imaging system (Bio-Rad). Quantification was based on relative band intensities. Indel percentage was determined by the formula, 100 × (1 − sqrt(1 − (b + c)/(a + b + c))), where a is the integrated intensity of the undigested PCR product, and b and c are the integrated intensities of each cleavage product.

Cells were grown aerobically overnight in LB broth at 37 °C. Supernatants were obtained by spinning down 2 ml of the cultures at 15,000 g for 10 min and filtered using a low protein-binding polyethersulfone (PES) membrane sterile filter (Foxx Life Sciences). 500 µl of the supernatant was then mixed with 2X Laemmli sample buffer (Bio-Rad, USA) and were boiled at 95 °C for 5 min. The samples were centrifuged and 10 µl of each supernatant was loaded onto a 4–20% stain-free polyacrylamide gel (Bio-Rad, USA). After electrophoresis, the gel was stained as per the recommendations of the Pierce silver stain kit (Thermo Scientific^TM, catalog #24600). Briefly, the gel was fixed in fixing solution (30% ethanol, 10% acetic acid). After ethanol and water wash, the gel was incubated in sensitizer working solution and then stained. After adding developer, the bands appear on the gel after 2-3 min. Stop solution (5% acetic acid) was used to terminate gel development and the gel was imaged using a GelDoc Gel imaging system (Bio-Rad, USA).