| Product Description | 2x Taq MasterMix is a premixed system composed of Taq DNA Polymerase, Mg2+, dNTPs, PCR stabilizers, and enhancers. The pre prepared PCR mixture makes the operation simpler and faster, and can minimize human error and contamination to the greatest extent possible. The original MasterMix formula results in high yield, strong repeatability, and good stability of amplified products. This product does not contain dyes. After the PCR program is completed, an appropriate amount of sample buffer can be added as needed for electrophoresis operation. The amplified PCR product has an "A" base attached to the 3 'end, making it suitable for direct use in T/A cloning. Mainly suitable for PCR amplification of DNA, DNA sequencing and other experiments. Quality control:
| T665627 | Component | 5ml | Storage | | T665627A | 2×Taq MasterMix | 5×1ml | -20℃. Avoid freeze/thaw cycle. | | T665627B | ddH₂O | 5×1ml | -20℃. Avoid freeze/thaw cycle. |
Notes: 2×Taq MasterMix contains Taq DNA Polymerase, 3mM MgCl2 and 400µM each dNTP
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After testing, there was no exogenous nuclease activity; PCR method for detecting residual DNA without host; Can effectively amplify single copy genes from multiple genomes.
Usage:
The following is an example of a PCR reaction system and reaction conditions for amplifying a 1 kb fragment using human genomic DNA as a template. In practical operation, corresponding improvements and optimizations should be made based on the template, primer structure, and target fragment size.
1. PCR reaction system
| Reagent | 50 μlReaction system | Final concentration | | 2×Taq MasterMix | 25 μl | 1× | | Forward Primer，10 µM | 2 μl | 0.4 μM | | Reverse Primer，10 µM | 2 μl | 0.4 μM | | Template DNA | <0.5 μg | <0.5 μg/50 μl | | ddH2O | up to 50 μl | / |
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Attention: The primer concentration should be between 0.1 and 1.0 as the final concentration μ M serves as a reference for setting the range. In the case of low amplification efficiency, the concentration of primers can be increased; When non-specific reactions occur, the primer concentration can be reduced to optimize the reaction system. 2. PCR reaction conditions | Step | Temperature | Time | / | | Pre denaturation | 95℃ | 2 min | / | | Denaturation | 94℃ | 30 s | 25-35 cycles | | Anneal | 55-65℃ | 30 s | 25-35 cycles | | Extend | 72℃ | 30 s | 25-35 cycles | | Finally extended | 72℃ | 2 min | / |
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Attention:
1) In general experiments, if the annealing temperature is 5 ℃ lower than the melting temperature Tm of the amplification primer, and the ideal amplification efficiency cannot be achieved, the annealing temperature should be appropriately reduced; When non-specific reactions occur, increase the annealing temperature to optimize the reaction conditions.
2) The extension time should be set according to the size of the amplified fragment. The amplification efficiency of Taq DNA Polymerase in this product is 2 kb/min.
3) The number of cycles can be set based on the downstream application of the amplification product. If the number of cycles is too small, the amplification amount is insufficient; If there are too many cycles, the probability of mismatches will increase, and non-specific backgrounds will be severe. So, while ensuring product yield, the number of cycles should be minimized as much as possible. |
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