2× Hieff Canace™ AdvanceFast High-Fidelity PCR Master Mix (With Dye) _ 10164ES
2× Hieff Canace™ AdvanceFast PCR Master Mix (With Dye) is a ready-to-use 2× pre-mixed solution containing Hieff Canace™ AdvanceFast High-Fidelity DNA Polymerase, dNTPs, and an opti mized buffer system, which contains pre-added electrophoresis indicators. The pre-mix contains pre-added electrophoresis indicator, PCR products can be directly electrophoresed, the amplification products are flat ends. 2× Hieff Canace™ AdvanceFast PCR Master Mix (With Dye) has the advantages of quick and easy, high sensitivity, high specificity, good stability, etc., the reaction system can be added with only the primers and templates. In addition, the product also contains a specific protective agent, so that the premix can still maintain stable activity after repeated freezing and thawing. Features Ultra-high fidelity: Low mismatch rate in mutation-prone high GC gene; Fast speed: The speed is as fast as 5 sec/kb; Wide applicability: It can be used to amplify genes with 20-80% GC content, and tolerate 20% blood and mouse lysates; Good stability: Stored at 37°C for 7 days without affecting performance; Easy use: Just adding primers and templates for amplification, with blue loading dye for direct electrophoresis. Specifications Cat.No. 10164ES01 10164ES03 10164ES08 Size 250 μL 1 mL 5×1 mL Figures 1. High fidelity Figure 1. Using genomes from different species as templates, six GC-rich fragments prone to base mutation/loss were selected for amplification. The respective PCR products were cloned into vectors, and 100 single clones for each sequence were picked for sequencing to determine base mismatch/mutation rates. The results show that 10164ES exhibits a lower base mismatch/mutation ratio (superior performance) compared to other brand reagents. 2. Extension time can be as short as 5 sec/kb for fragments under 10 kb Figure 2. Fragments of varying lengths (0.4–10 kb) were amplified from 100 ng of human genomic DNA template using 10164ES and different products, with the extension time set to 5 sec/kb for all. The results show that 10164ES can successfully amplify fragments under 10 kb at an extension speed of 5 sec/kb. Marker: Yeasen 10510ES. 3. Capable of amplifying gene fragments with varying GC contents Figure 3. Fragments with 23–76% GC content were amplified from human gDNA templates using 10164ES, with the extension time set to 5 sec/kb. The results show that the product exhibits good amplification specificity and is compatible with the amplification of fragments with varying GC contents. Marker: Yeasen 10510ES. 4. Compatible with various types of templates Figure 4. Fragments of different lengths were amplified from various samples using 10164ES, with the extension time set to 5–10 sec/kb. The results show that the product exhibits good amplification specificity and is compatible with the amplification of different fragments from various templates. 1. Mouse gDNA 1 kb; 2. Mouse gDNA 2 kb; 3. Mouse gDNA 6 kb; 4. Corn gDNA 1 kb; 5. Rice gDNA 2 kb-A; 6. Rice gDNA 2 kb-B; 7. Human 293 cell cDNA 1 kb; 8. Human 293 cell cDNA 2 kb; 9. Human 293 cell cDNA 3 kb; 10. Human 293 cell cDNA 12 kb; 11. E. coli culture 8 kb; 12. E. coli culture 16 kb; 13. Rice gDNA 10 kb-A; 14. Rice gDNA 10 kb-B; 15. Rice gDNA 10 kb-C; 16. Rice gDNA 20 kb-A; 17. Rice gDNA 20 kb-B; 18. Direct amplification from human blood 3 kb. Marker: Yeasen 10510ES. 5. High detection rate Figure 5. Fragments of 0.5–10 kb with varying GC contents from Arabidopsis thaliana, Brassica napus, and Solanum tuberosum were amplified using 10164ES with an extension time of 5 sec/kb. The results show that 10164ES ensures successful amplification. Marker: Yeasen 10510ES. Figure 6. Fragments of 0.5–10 kb covering 20–80% GC content with randomly selected sequences were amplified from Arabidopsis thaliana, Brassica napus, Drosophila, Caenorhabditis elegans, and zebrafish using 10164ES, with an extension time of 5 sec/kb. The results show that 10164ES demonstrates a favorable detection rate. 6. Excellent Tolerance Figure 7. Amplification of different-sized fragments from blood and mouse lysate using 10164ES. The results show that 10164ES can tolerate 1–20% blood and exhibits a certain resistance to inhibition from mouse lysate. M: Yeasen 10510ES. Storage This product should be stored at -25~-15℃ for 1 years. Instructions Recommended PCR reaction systems. Components Volume（μL） Final concentration 2× Hieff Canace™ AdvanceFast PCR Master Mix (With Dye)* 25 1× Template** x - Forward Primer（10 μmol/L）*** 2 0.4 μmol/L Reverse Primer（10 μmol/L） 2 0.4 μmol/L ddH2O Up to 50 - Table 1 PCR reaction system *In 1× premixes containing 2 mM Mg2+ and 200 μM dNTPs. **Recommended range 10-200 ng, cDNA sample upload volume range not more than 1/10 of the reaction system, recommended 1-2.5 μL. ***The final primer concentration in the PCR reaction system ranges from 0.2-1 μM, and 0.4 μM is recommended. Reaction program. Cycle step Temp. Time Cycles Initial denaturation 98℃ 30 sec 1 Denaturation 98℃ 10 sec 30-35 Annealing* 60℃ 5 sec Extension** 72℃ 5-10 sec/kb Final extension 72℃ 2 min 1 Table 2 PCR reaction program *Recommended temperature: 60°C, a temperature gradient can be set up to find the optimal temperature for primer annealing. The recommended annealing time is set to 5 sec and can be adjusted from 5-30 sec. Too long annealing time may result in diffuse amplification products on the gel. **Extension time: Recommended 5 sec/kb, can also be extended to 10 sec/kb as needed. Notes This product is for research use only. Please operate with lab coats and disposable gloves，for your safety. FAQ Q: What are the differences between 10163ES and 10164ES? A: 10164ES is a reagent for premixed loading dye. Users don't need to purchase the loading dye and spend time adding the dye one well at a time during the loading and electrophoresis process. b. 10163ES is the colorless version, which does not contain dye. It won't cause interference for users' subsequent enzymatic digestion and other experiments, ensuring efficient enzymatic digestion in the future. Q: Do the amplification products of 10163ES and 10164ES have an A tail? A: Does not end with A. The product is a blunt-end product, which can be used for subsequent homologous recombination cloning (10911\10922ES) or TOPO blunt-end cloning (10906\10909ES). If TA cloning is to be performed later, the user will need to purchase dATP (10118ES) and regular Taq enzyme (10101ES) separately. Documents: Safety Data Sheet 10164_MSDS_HB251118.PDF Manuals 10164ES-Ver. 20230720.pdf Citations & References: [1] Dong W, Zhu Y, Chang H, et al. An SHR-SCR module specifies legume cortical cell fate to enable nodulation. Nature. 2021;589(7843):586-590. doi:10.1038/s41586-020-3016-z(IF:42.779) [2] Sun L, Yan Y, Lv H, et al. Rapamycin targets STAT3 and impacts c-Myc to suppress tumor growth. Cell Chem Biol. 2022;29(3):373-385.e6. doi:10.1016/j.chembiol.2021.10.006(IF:8.116) [3] Ye M, Xiong L, Dong Y, et al. The Potential Role of the Methionine Aminopeptidase Gene PxMetAP1 in a Cosmopolitan Pest for Bacillus thuringiensis Toxin Tolerance. Int J Mol Sci. 2022;23(21):13005. 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