Description
Product description
2× HotStart PCR Master Mix contains heat-stabilized Taq DNA Polymerase modified with antibodies, adds strong elongation factor and optimized buffer system, and has super high amplification efficiency. It is very suitable for PCR amplification of most colonies such as E. coli, Agrobacterium, and yeast. The extension speed of amplification of complex templates within 3 kb can reach 1 sec/kb, 3-6 kb to 3 sec/kb, 6-10 kb to 5 sec/kb and 10 kb to 10 sec/kb; the amplification speed of simple templates such as plasmid within 6 kb can reach 1 sec/kb, which can greatly save PCR reaction time. Meanwhile, Mix contains dNTPs, Mg2+, and it can be amplified only with adding primers and template. In addition, Mix contains red tracer dye, which can be used by electrophoresis directly after the end of the reaction. The protective agent added to the system enables the product to maintain the stable activity after repeated freezing and thawing. The 3′ end of the PCR product bands A and can be easily cloned into the T vector.
Components
|
Components No.
|
N132001E
|
N132001S
|
N132001M
|
N132001L
|
|
Size
|
1 mL
|
5×1 mL
|
50×1 mL
|
100×1 mL
|
Specifications
|
Product specification
|
Master Mix
|
|
Concentration
|
2×
|
|
Hot Start
|
Built-in Hot Start
|
|
Overhang
|
3 '-A
|
|
Reaction speed
|
Rapid
|
|
Size (Final Product)
|
Up to 15 kb
|
|
Conditions for transportation
|
Dry ice
|
Shipping and Storage
Dry ice shipping. -15℃ ~ -25℃ storage, valid for two years.
Notes
1. For your safety and health, please wear lab coats and disposable gloves for operation.
2. This product is for research use ONLY!
Instructions
1. Reaction System
Table 1 Reaction system
|
Components
|
Size (μL)
|
Size (μL)
|
Final concentration
|
|
2×HotStart Fast PCR Master Mix (With Dye)*
|
25
|
12.5
|
1×
|
|
Template DNA**
|
suitable
|
suitable
|
-
|
|
Forward primer (10 μmol/L)
|
2
|
1
|
0.4-0.5 μM
|
|
Reverse primer (10 μmol/L）
|
2
|
1
|
0.4-0.5 μM
|
|
ddH2O
|
to 50
|
to 25
|
-
|
【Note】:
1) *The 2×Mix contains 2 mM Mg2+ and 200 μM dNTPs, thawed thoroughly before use.
2) **E. coli and Agrobacterium can directly absorb bacterial liquid or pick bacteria samples; It is recommended that the yeast liquid and
3) **Strain be boiled for 5 min, then put into the -80℃ refrigerator for 3 min, and then serve the sample after thawing. Note that the bacterial liquid sample should be shaken and mixed before sampling, in which the recommended amount of bacterial liquid sample is 2-4 μL (0.5-0.8 OD600).
Recommended usage of the different templates:
Table 2 Recommended usage of different templates
|
Type of template
|
Segment usage range (25 μL reaction system)
|
|
Genomic DNA
|
10–1,000 ng
|
|
Plasmid or λDNA
|
0.5-50 ng
|
|
E. coli bacteria solution
|
0.5-0.8 OD600
|
【Note】:
***The range of final primer concentration in PCR reaction system is 0.2-1 μM, and 0.4 μM is recommended.
2. Amplification Protocol
Table 3 Amplification protocol
|
Cycle steps
|
Temperature (°C)
|
Time
|
Cycles
|
|
Predenaturation
|
95
|
3 min
|
1
|
|
Denaturation
|
95
|
15 sec
|
30-35
|
|
Annealing*
|
60
|
20 sec
|
|
Extension**
|
72
|
1-10 sec/kb
|
|
Final extension
|
72
|
5 min
|
1
|
【Note】:
a. *Recommended annealing temperature: 60°C, you can also set up a temperature gradient according to your own needs to explore the optimal temperature for primer annealing. The recommended annealing time is set to 20 sec, which can be adjusted within 10-30 sec. Too long annealing time may lead to dispersion of the amplified product on the glue.
b. **Extension speed: 1sec/kbfor complex templates such as genomes and E. coli within 3 kb, 3 sec/kb for complex templates within 6 kb, 5 sec/kb for most complex templates within 10 kb, and 10 sec/kb for complex template fragments over 10 kb. For simple templates, such as plasmids less than 6 kb, set 1 sec/kb; for simple templates, such as 6-10 kb plasmids, set 3 sec/kb; and for simple templates, such as plasmids larger than 10 kb, set 5-10 sec/kb. If it is necessary to increase production, the extension time can be extended appropriately, and should not exceed 30 sec/kb.
