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Description
2 × Taq Master MixProduct Specification Physical Appearance Liquid Storage Buffer 20 mM Tris HCl (pH 8. 6 @ 25C), 100 mM KCl, 3 mM MgCl2, 0. 1% Tween 20, 0. 1% IGEPAL CA 630, 10% Glycerol Stability & Storage Store at 25 ~ 15 for 2 years Reference 1. Lawyer F C,Stoffel S,Saiki R K ,et al. Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus.[J]. Journal of Biological Chemistry, 1989, 264. 2. Eom S H,Steitz T
Product Specification
| Physical Appearance | Liquid |
| Storage Buffer | 20 mM Tris-HCl (pH 8.6 @ 25°C), 100 mM KCl, 3 mM MgCl2, 0.1% Tween® 20, 0.1% IGEPAL® CA-630, 10% Glycerol |
| Stability & Storage | Store at -25 ~ -15℃ for 2 years |
| Reference | 1. Lawyer F C,Stoffel S,Saiki R K ,et al. Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus.[J].Journal of Biological Chemistry, 1989, 264. |
Components
Protocol
1. After thawing, gently rotate and briefly centrifuge.
2. Prepare the reaction system according to the table:
Component |
Volume |
2 × Taq Master Mix |
25 μl |
Forward Primer (10 μM) |
2 μl |
Reverse Primer (10 μM) |
2 μl |
Template DNA* |
x μl |
RNase Free dH2O |
Up to 50 μl |
*Different templates have different optimal reaction concentrations, and the following table shows the recommended amount of template for the 50 μl reaction system:
Template type |
Template quantity |
Animal and plant genomic DNA |
0.1 - 1 μg |
Escherichia coli genomic DNA |
10 - 100 ng |
cDNA |
1 - 5 μl (No more than 1/10 of the total volume of the PCR reaction) |
Plasmid DNA |
0.1 - 10 ng |
λ DNA |
0.5 - 10 ng |
3. Gently mix and briefly centrifuge.
4. Prepare the PCR reaction system according to the table:
Step |
Temperature |
Time |
Number of Cycles |
Initial Denaturation |
94°C |
3 minutes |
1 cycle |
|
Denaturation Annealing** Extension |
94°C 50-60°C 72°C |
30 seconds 30 seconds 1 min/kb |
25–35 cycles
|
Final Extension |
72°C |
10 minutes |
1 cycle |
Soak |
4°C |
Indefinite |
1 cycle |
* The preconditioning condition is suitable for the vast majority of amplification reactions and can be adjusted according to the complexity of the template. If the template structure is complex, the preconditioning time can be extended to 5-10 minutes to improve the preconditioning effect.
** The annealing temperature needs to be adjusted according to the Tm value of the primer, generally set at 3 to 5 ℃ lower than the Tm value of the primer; for complex templates, the annealing temperature needs to be adjusted and the extension time extended to achieve efficient amplification.
Guidelines
1. It needs to be dissolved completely before use to prevent uneven ion concentration.
2. The number of cycles should be selected according to the purpose of the experiment. If the number of cycles is too small, it will lead to insufficient amplification; if the number of cycles is too large, the amplification will increase, but the mutation rate will also increase, and non-specific amplification will occur.
3. Set the appropriate annealing temperature according to the Tm value of the primer. If the annealing temperature is too low, it will cause non-specific amplification; if it is too high, it may not be amplified.
4. Because Taq DNA Polymerase also has certain reaction activity at room temperature, the PCR reaction system should be prepared on ice and then placed in the PCR instrument for reaction. This can reduce non-specific amplification that occurs during the preparation of the reaction and help obtain high specificity amplification results.
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