2× HiFi PCR Mix for NGS HYA446
HiFi DNA polymerase is a typical B-family polymerase with a unique structure. It is a new enzyme similar to Pyrococcus furiosus, fused with a continuous synthesis enhancement domain, which improves elongation, yield and specificity. HiFi DNA polymerase is equipped with specific antibodies, which effectively inhibits non-specific amplification and improves the stability of HiFi DNA polymerase. HiFi DNA polymerase has 5'-3' continuous synthesis activity and 3'-5' exonuclease activity, and its amplified products are blunt-ended. HiFi DNA polymerase is a mutant polymerase modified by genetic engineering. It has the ability to incorporate dUTP and can also amplify complex templates with dU. 2× HiFi PCR Mix for NGS is an easy-to-operate 2X Mix, which contains 2× HiFi PCR Mix, buffer, Mg+ and dNTPs, etc. Only additional DNA template, primers and water are needed in the amplification system. Perform amplification. All batches of 2× HiFi PCR Mix for NGS have undergone strict quality control to ensure the stable performance of each batch of reagents.
Components
|
Components |
Size-1 |
Size-2 |
|
2× HiFi PCR Mix for NGS |
24T |
96T |
Storage
Store at -20℃.
Reaction System
|
Components |
Volume |
|
2× HiFi PCR Mix for NGS |
25 uL |
|
Template DNA* |
X uL |
|
Forward primer (20 µM )** |
2.5 uL |
|
Reverse primer (20 µM ) ** |
2.5 uL |
|
Nuclease-free Water |
Up to 50 uL |
*The DNA template refers to the purified product of the linker connected to the magnetic beads during the library construction process.
**The primers are the library primers used in the process of NGS library construction.
Reaction Conditions
|
Step |
Temperature |
Time |
Cycles |
|
Pre Denaturation |
98 ℃ |
45s |
1 |
|
Denaturation |
98 ℃ |
15 s |
Recommended number of cycles based on input* |
|
Annealing |
60 ℃ |
30 s |
|
|
Extension |
72℃ |
30 s |
|
|
Hold |
72℃ 4-10℃ |
1 min ∞ |
1 |
*Recommended number of cycles based on input：
|
Input DNA(fragmented DNA) |
Number of PCR cycles |
|
1ng |
13-15 |
|
10ng |
9-11 |
|
25ng |
7-9 |
|
50ng |
6-7 |
|
100ng |
5-6 |
|
250ng |
4-5 |
|
500ng |
3-4 |
|
1000ng |
2-3 |
Notes
1. It is recommended to prepare all reaction components on ice, because the 3'-5' exonuclease activity of 2× HiFi PCR Mix for NGS at room temperature will cause primer degradation;
2. All components should be mixed well and centrifuged instantaneously before use.
3. The polymerase is specially developed for the NGS library preparation and amplification system.
4. For cloning and other aspects of amplification, please select other molecular amplification enzymes.