Taq DNA Polymerase is a recombinant enzyme expressed and purified by Escherichia coli. Its gene is derived from the Thermus aquaticus polymer. The protein has a molecular weight of 94 kDa and exhibits 5 '→ 3' DNA polymerase activity and 5 '→ 3' exonuclease activity, without 3 '→ 5' exonuclease activity. The enzyme has an elongation rate of 2 kb/min and can amplify fragments up to 5 kb in length. The amplified PCR product has an "A" base attached to its 3 'end, making it suitable for direct use in T/A cloning. This product has the characteristics of fast extension speed and high amplification efficiency, and is mainly suitable for PCR amplification of DNA fragments, DNA sequence determination and other experiments.
| T665586 | Component | 500 U | 2500 U | 10 KU | Storage | | T665586A | Taq DNA Polymerase, 5 U/μL | 100 μL | 5×100 μL | 2×1 mL | -20℃. Avoid freeze/thaw cycle. | | T665586B | 10×PCR Buffer | 1.8 mL | 5×1.8 mL | 8×5 mL | -20℃. Avoid freeze/thaw cycle. |
Notes: 10×PCR Buffer contains 15mM Mg2+. |
Activity definition:
Using activated salmon sperm DNA as a template/primer, the amount of enzyme required to incorporate 10 nmol of deoxyribonucleotide into acidic insoluble substances is defined as 1 active unit (U) at 74 ℃ for 30 minutes.
Quality control:
After multiple column purifications, SDS-PAGE detected a purity of over 99%; No exogenous nuclease activity detected; PCR method for detecting residual DNA without host; Can effectively amplify single copy genes in the human genome; Store at room temperature for one month without significant changes in activity.
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 | | 10×PCR Buffer | 5 μl | 1× | | dNTP Mix，10 mM each | 1 μl | 200 μM each | | 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 | | Taq DNA Polymerase，5 U/μl | 0.25-0.5 μl | 1.25-2.5 U/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 | 94℃ | 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.
