2 Contents I. Kit Contents and Storage II. Product Overview III. Methods Experimental Outline Genomic DNA Preparation Obtain Hybrid DNA Digest Hybrid DNA and Gel Analysis
3 I Kit Contents and Storage Table1. The Components of Knockout Detection (KOD) Kit Description 25 rxns 10 x G-Taq Buffer 80 μl G-Taq DNA polymerase 8 μl 5 x Detection Buffer 100 μl KOD enzyme Positive control DNA (100ng/μl) 25 μl 30 μl 6 x Stop Buffer 100 μl Reagent A 6 ml x 1 Reagent B 300 μl x 1 Storage Store G-Taq DNA polymerase, KOD enzyme and positive control DNA at -20 C; store 5 x detection buffer, 10 x G-Taq buffer, 6 x stop buffer, reagent A and reagent B at 2-8 C. Note: KOD enzyme is solid at -20 C. Upon receiving, we recommend to thaw it at 4 C and store in aliquots to avoid repeated freeze-thaw cycles.
4 II Product Overview Knockout Detection (K OD) Kit provides a simple and highly effective method to detect gene knockout and gene polymorphism. The core component of the kit is the knockout detection (KOD) enzyme, which has strong endonuclease activity and is homologous to Cel. The KOD enzyme can efficiently recognize and cleave the mismatches in heterogeneous DNA duplexes. Our KOD kit can be widely used for gene knockout detection in mammals, bacteria, fungi, virus and plants, especially applicable to screen positive clones and detect knockout efficiency using CRISPR/Cas9, TALENs or ZENs technologies. Product features Fast 5-20 min of digestion time is enough to detect any type of mutations such as substitution, insertion and deletion. Simple PCR product can be directly used for enzyme digestion without purification. Efficient The detectable mutation size ranges from 1 bp to several hundreds of base pairs. Convenient The digested PCR product can be analyzed by regular agarose gel electrophoresis. Mixed-sample analysis Enzyme digestion of PCR product from cell pool can be used to calculate mutation efficiency. III Methods Experimental Outline
5 Genomic DNA Preparation Only cells are required to isolate genomic DNA using reagent A and reagent B. 1) Harvest cells by centrifugation at 1500 rpm, 4 C for 5 min and carefully remove supernatant. 2) Wash cells with 150 μl cold PBS, centrifuge, and discard supernatant. Repeat this step once if large amount of cells are harvested. 3) Add μl premixed reagent A and reagent B solution #, mix to lyse cells and then incubate on ice for 10 min. 4) Add 2 vol. pure ethanol, mix, and incubate at -20 C for at least 20 min. 5) Centrifuge at 1500 rpm, 4 C for min, and discard supernatant. 6) Wash twice with μl pre-cold 75% ethanol, centrifuge at 1500 rpm, 4 C for 10 min, and discard supernatant. 7) Air dry and dissolve DNA in μl H 2 O. # Mix reagent A and reagent B at 20:1 and prepare freshly before use. Obtain Hybrid DNA Target region is amplified by PCR with specific primers. The PCR reaction is as followed: Genomic DNA 100 ng 10 G-Taq buffer 3 μl DMSO 1.8 μl dntp (10 mm each) 0.6 μl Detect-PrimerF (10 μm) 1.2 μl Detect-PrimerR (10 μm) 1.2 μl G-Taq DNA polymerase 0.3 μl Add ddh 2 O to 30 μl Obtain hybrid DNA: incubate PCR product at 98 C for 3 min, and then cool down to 40 C at room temperature.
6 Digest Hybrid DNA and Gel Analysis Annealed PCR products 2-5 μl 5 Detection buffer 2 μl Detection enzyme 1 μl Add ddh 2 O to 10 μl Incubate at 45 C for min (no longer that 30 min), and then add 2μl 6 stop buffer. Figure 1. Example of 2% TAE-agarose gel analysis of digestion products M: 100-bp DNA Marker positive: positive control DNA 1-7: sample 1-7 Hybrid DNA is used as positive control. Two bands (134bp and 259bp) indicate the cleaved fragments from heteroduplex DNA and one 393bp-band indicates the homo-duplex DNA fragment which cannot be cleaved. Sample 3, 5, and 6 are potential positive clones which can be selected for subsequent verification by TA cloning and sequencing.