Off-target sites were predicted utilizing the Optimized CRISPR Design (http://crispr.mit.edu , F. Zhang laboratory, MIT 2015). Loci of predicted sites (see Supplementary Table S2 ) were PCR amplified from gDNA of PLB-985 WT and PLB-985 NCF1 ΔGT cells. Corresponding PCR amplification products were mixed in a ratio of 1:1, while PCR amplification product of PLB-985 WT was used as a control. The samples were denatured at 95 °C for 10 minutes, slowly renatured, and digested using Surveyor® Mutation Detection Kit For Standard Gel Electrophoresis (Integrated DNA Technologies, Leuven, Belgium) according to the manufacturer’s instructions.
Surveyor mutation detection kit for standard gel electrophoresis
Manufactured by Integrated DNA Technologies
Sourced in Belgium
The Surveyor® Mutation Detection Kit for Standard Gel Electrophoresis is a laboratory product designed to detect DNA sequence variations. It enables the identification of single nucleotide polymorphisms (SNPs) and small insertions or deletions (indels) through the analysis of DNA samples using standard gel electrophoresis.
Automatically generated - may contain errors
5 protocols using surveyor mutation detection kit for standard gel electrophoresis
1
CRISPR Off-Target Site Analysis
2
Surveyor Assay for Genome Editing
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Genomic DNA editing was detected using a Surveyor Mutation Detection Kit for Standard Gel Electrophoresis (Integrated DNA Technologies) according to the manufacturer's instructions. Briefly, genomic DNA was purified, followed by PCR, and PCR fragments were extracted. DNA was annealed in 1× prime star buffer (Takara) and the surveyor enzyme was added to digest the annealed DNA fragments, followed with gel electrophoresis to detect the DNA bands after digestion.
3
Detecting Genomic DNA Editing
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Genomic DNA editing was detected using Surveyor® Mutation Detection Kit for Standard Gel Electrophoresis (Integrated DNA Technologies) according to manufacturer’s instructions. Briefly, genomic DNA was extracted by DNeasy Blood & Tissue Kit (Qiagen). PCR fragments were purified by GeneJET PCR Purification Kit (K0701). After that, PCR fragments were annealing in 1× prime star buffer (Takara) and Surveyor enzyme was added to digest the annealed DNA fragments at 42 °C for 1 h. Gel Electrophoresis on agarose gel was used to detect the digestion.
4
PCR-Based CRISPR Mutation Detection
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The primer sets for PCR were designed to cover all possible mutation sites (Fig. 1B ). PCR was performed using the Expand High FidelityPLUS PCR System (Roche). The PCR amplification procedure was as follows: initial denaturation for 3 min at 94 °C; followed by 34 cycles of 94 °C for 30 s, 60 °C for 45 s, and 72 °C for 45 s; and a final elongation at 72 °C for 10 min. The resulting PCR product length was verified in a 1.5% agarose gel. The Surveyor® mutation detection kit for standard gel electrophoresis (Integrated DNA Technologies, Coralville, IA) was used to detect mutations81 , 82 (link); PCR products were denatured and re-annealed as follows: 95 °C for 10 min; 95 to 85 °C at −2 °C/s; 85 to 35 °C at −0.3 °C/s; cooling to 4 °C; and then Nuclease S was mixed with Enhancer S and MgCl2 and added to the PCR products above and incubated at 42 °C for 1 hour. The digested products were separated in a 2% agarose gel and compared to those from a non-edited channel catfish (Fig. 3A ).
5
Surveyor Assay for Genomic Editing
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Genomic DNA editing was detected using Surveyor® Mutation Detection Kit for Standard Gel Electrophoresis (Integrated DNA Technologies) according to manufacturer's instructions. Briefly, genomic DNA was extracted by DNeasy Blood & Tissue Kit (QIAGEN). PCR fragments were purified by GeneJET PCR Purification Kit (K0701). After that, PCR fragments were annealing in 1X prime star buffer (Takara) and Surveyor enzyme was added to digest the annealed DNA fragments at 42 o C for 1h. Gel Electrophoresis on agarose gel was used to detect the digestion.
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