2×A9 LongHiFi PCR MasterMix(With Dye)
Lot # Check on the product label
Size: 5 ml
Storage and Expiration: Store at -20°C for 2 years.
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Price: $280.00
Catalog# BPC1020-2
Lot # Check on the product label
Size: 5 ml
Storage and Expiration: Store at -20°C for 2 years.
Description: This product contains 2×A9 LongHiFi PCR MasterMix, dNTPs and optimized reaction buffer at a concentration of 2×. When using, just add template, primer, and make up water to the final concentration of Mix to 1×. A9 is a mixture of a variety of ultra-fidelity enzymes modified by the most advanced genetic engineering with extension factors, which greatly improves the amplification length, amplification speed, fidelity and yield. A9 Mix is the first choice for ultra-fidelity and rapid amplification of long fragments. This product contains a red tracer dye. It can be directly loaded for electrophoresis without adding a loading buffer; it can also be purified for subsequent operations such as restriction digestion, ligation, and fluorescence sequencing.
Recommended PCR system settings
|
Component |
25 µl reaction |
50 µl reaction |
Final Concentration |
|
2× A9 PCR MasterMix |
12.5 μl |
25 μl |
1 × |
|
Forward Primer(10 μM) |
0.5 μl |
1 µl |
0.2 μM |
|
Reverse Primer(10 μM) |
0.5 μl |
1 μl |
0.2 μM |
|
Template DNA |
as required |
as required |
|
|
ddH2O |
up to 25 μl |
up to 50 μl |
|
Reference template dosage (50 μl reaction system):
Plasmid: 0.1-10ng; bacterial genome: 10-100ng; human genome: 50-150ng; cDNA: 1-5μl from RT reaction.
Recommended PCR cycling conditions:
|
Step |
Temperature |
Time |
Cycle Number |
|
Initial denaturation |
95°C |
3 minutes |
|
|
Denaturation |
95°C |
10 seconds |
30 cycles |
|
Annealing |
55°C |
10-15 seconds |
|
|
Extension |
72°C |
15-20 seconds / kb |
|
|
Final Extension |
72°C |
2-5 minutes |
|
|
|
4-8°C |
Hold |
|
Precautions:
1. The amplification speed of A8 is fast. Simple templates such as plasmids and simple genomes can be used at 10-15 seconds/kb; complex templates such as human genomes can be used at 20-25 seconds/kb.
2. If the electrophoresis finds that the main band is smeared up and down, or the smearing band dragged down from the sample hole in the swimming lane, it generally indicates that the extension time is too long.
3. Longer, the gradient shortens the extension time (10-15 seconds/kb is recommended for the gradient to reduce the extension time) until a satisfactory result is obtained.
4. For templates with high GC content, the pre-denaturation and denaturation temperature can be increased to 98°C. A9 has strong heat resistance, 98°C does not change the activity of A9.If the GC content of the amplified template is high or the template is complex and the amplification effect is not good, can add DMSO to the reaction mixture to a final concentration of 1%-8%, and follow a 1% gradient increase to find the optimal concentration. Or add betaine to a final concentration of 1.0-1.7 M. And use Touchdown PCR.
