2×A9 LongHiFi PCR MasterMix(With Dye)
Lot # Check on the product label
Size: 1 ml
Storage and Expiration: Store at -20°C for 2 years.
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Price: $70.00
Catalog# BPC1020-1
Lot # Check on the product label
Size: 1 ml
Storage and Expiration: Store at -20°C for 2 years.
Description
This product contains A9 LongHiFi DNA Polymerase, dNTPs and an optimised reaction buffer with concentration of 2×. When use, only add template and primers and make up the water to a final Mix concentration of 1×. A9 Mix is a blend of ultra-fidelity enzymes and extension factors using state-of-the-art genetic engineering to greatly improve amplification length, speed, fidelity and yield.
A9 Mix is the top choice for fast, ultra-fidelity amplification of long fragments. This product contains red tracer dye and can be used directly for electrophoresis without the addition of loading buffer; it can also be purified for subsequent operations such as enzyme digestion, ligation and fluorometric sequencing.
1. Fast amplification speed: the extension speed can reach 3-4kb/min, which is 6-8 times faster than pfu.
2. High amplification yield: the general PCR product volume is 50%-100% higher than the traditional pfu yield.
3. Excellent fidelity: the fidelity is more than 54 folds that of taq. Generally a randomly selected bacterium is sequenced as a correct mutation-free colony.
4. Long amplification lengths: using complex genomic DNA as a template is suitable for amplifying products up to about 10kb, using simple genomic, plasmid and phage DNA as a template is suitable for amplifying products up to about 15kb.
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: 0.1-10 ng; bacterial genome: 10-100 ng; human genome: 50-150 ng; cDNA: 1-5 μl 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. A9 amplification is fast. Simple templates such as plasmids and simple genomes can be amplified in 15-20 seconds/kb; complex templates such as human genomes can be amplified in 25-30 seconds/kb.
2. If the electrophoresis reveals blurred trailing on the main band or a smear band trailing down from the upper sample well, it is usually indicated that the extension time is too long, so shorten the extension time in a gradient (10-15 sec/kb is recommended) until satisfactory results are obtained.
3. For templates with high GC content, the pre-denaturation and denaturation temperature can be increased to 98 oC. A9 is highly heat resistant and 98 oC does not change the activity of A9.
4. If the GC content of the amplification template is high or the template is complex, DMSO can be added to the reaction mixture to a final concentration of 1%-8%, increasing in a 1% gradient to find the optimum concentration. Alternatively, add betaine to a final concentration of 1.0-1.7 M and use landing PCR (Touchdown PCR).
