Hotstart PCR Master Mix HYA444
Hotstart PCR Master Mix is a universal PCR amplification product developed by our company. This product uses a hot-start rapid amplification enzyme, with the fastest amplification rate reaching 1 kb/10 sec and higher extension efficiency, which can be applied to rapid PCR reactions. Moreover, it has good resistance to PCR amplification inhibitors, making it particularly suitable for rapid high-throughput detection.
Components
2× Hotstart PCR Master Mix
*This reagent already contains components such as hot-start enzyme, PCR Buffer, dNTPs, MgCl₂, stabilizers, and enhancers.
Storage
Long-term storage at -20°C; can be stored at 4°C for 3 months. Mix well before use and avoid repeated freeze-thaw cycles.
Reaction System
|
Components |
25uL Reaction System |
50uL Reaction System |
|
2× Hotstart PCR Master Mix |
12.5uL |
25uL |
|
25×PrimerMix* |
1uL |
2uL |
|
Template** |
-- |
-- |
|
ddH2O |
Up to 25uL |
Up to 50uL |
*A final primer concentration of 0.2 μM typically yields optimal results.If amplification efficiency is low, adjust primer concentration within 0.2–1.0 μM.
**The copy number of target genes varies among different types of templates. Perform serial dilutions to determine the optimal template input volume for your specific sample.
Reaction Conditions
|
Standard PCR Protocol |
|||
|
Step |
Temperature |
Time |
Cycles |
|
Digestion |
50℃ |
2 min |
1 |
|
Hot Start |
95 ℃ |
1-5 min |
1 |
|
Denaturation |
95 ℃ |
10-20 s |
30 |
|
Annealing |
55~65℃ |
10-30 s |
|
|
Extension |
72℃ |
x s* |
|
|
Rapid PCR Protocol |
|||
|
Step |
Temperature |
Time |
Cycles |
|
Digestion |
50℃ |
2 min |
1 |
|
Hot Start |
95 ℃ |
1-2 min |
1 |
|
Denaturation |
95 ℃ |
10 s |
25-30 |
|
Annealing and Extension |
55~65℃ |
x s* |
|
*It is recommended to calculate based on the maximum fragment size of 10~20sec/kb.
Quality Control
1. Functional Testing:PCR sensitivity, specificity, and reproducibility.
2. Free of exogenous nuclease activity; no contamination by exogenous endonucleases or exonucleases.
Technical description
1. Use 95℃ or 94℃ 1~5min hot start;
2. The amplification rate of fast DNA polymerase can reach up to 1kb/10s.
3. The system has strong adaptability and is suitable for fast amplification reactions.
4. Fast DNA polymerase has 5'-3' polymerase and 5'-3' exonuclease activity; no 3'-5' exonuclease activity and no proofreading function.
5. Suitable for ordinary PCR, gene chip and other detection methods.
6. The 3' end of the PCR product is A, and the product can be directly cloned into T vector.
7. If the amplification efficiency is not enough when setting 10sec/kb extension, it can be extended to 20sec/kb or longer extension time.
8. When the crude sample is not amplified well, the template can be appropriately diluted and then amplified.