Nucleic acid polymerase chain reaction optimized expanding method based on nano metal silver
A technology of nano-metallic silver and nucleic acid polymerase, which is applied in biochemical equipment and methods, microbial measurement/inspection, fermentation, etc. It can solve the problems of complex extraction and purification of SSB protein technology, expensive commercial kits, and short time limit , to achieve the effect of convenient processing and application, simple optimization method, easy to grasp, and easy to save
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Embodiment 1
[0046] The optimization of silver nanopowder to PCR reaction is aimed at the optimization and improvement of some PCR amplifications that form non-specific amplification, and amplifies long fragments with a length of 4249bp, including the following steps:
[0047] 1. Preparation of nanometer metal silver (silver nanopowder) suspension:
[0048] The silver nanopowder was purchased from Sigma, and the nano-metallic silver suspension solution with a concentration of (W / V) 10mg / mL was prepared. First, 10mg of sterilized nano-metallic silver was accurately weighed and placed in a sterilized 1.5mL small centrifuge tube. , slowly add sterilized water with a pipette to a volume of 1 mL, and ultrasonically suspend at 40 KHz for 10 min.
[0049] 2. The configuration of the PCR system, the specific composition is as follows:
[0050] Taq enzyme (5U / μL)
0.45 μL
Pfu enzyme (2.5U / μL)
0.45 μL
10×PCR buffer
2.5μL
dNTP substrate (2.5mM)
3.5μL
...
Embodiment 2
[0060] The optimization of nano-metallic silver on the PCR amplification of ultra-long fragments, the optimization and improvement of PCR amplification of ultra-long fragments, the amplification of long fragments with a length of 15000bp, including the following steps:
[0061] 1. Preparation of nanometer metal silver (silver nanopowder) suspension:
[0062] With embodiment 1.
[0063] 2. The configuration of the PCR system, the specific composition is as follows:
[0064] Taq enzyme (5U / μL)
0.45 μL
Pfu enzyme (2.5U / μL)
0.45 μL
10×PCR buffer
2.5μL
dNTP substrate (2.5mM)
3.5μL
Mg 2+ (25mM)
1.75μL
Primer 1 (1 μM)
3.75 μL
Primer 2 (1 μM)
3.75 μL
template
1μL
[0065] Among them, the template is λDNA, which was purchased from Sanbo Polygala Bioengineering Company, and Taq enzyme was purchased from Sanbo Polygala Bioengineering Company.
[0066] A total of 3 PCR system tubes were co...
Embodiment 3
[0074] Silver nanopowder optimizes high GC content PCR amplification, optimizes and improves high GC content amplification, and amplifies DNA molecules with a length of 2872bp, including the following steps:
[0075] 1. Preparation of nanometer metal silver (silver nanopowder) suspension:
[0076] With embodiment 1.
[0077] 2. The configuration of the PCR system, the specific composition is as follows:
[0078] Taq enzyme (5U / μL)
0.45 μL
Pfu enzyme (2.5U / μL)
0.45 μL
[0079] 10×PCR buffer
2.5μL
dNTP substrate (2.5mM)
3.5μL
Mg 2+ (25mM)
1.75μL
Primer 1 (1 μL)
3.75 μL
Primer 2 (1 μM)
3.75 μL
template
1μL
[0080] Among them, the template is λDNA, which was purchased from Sanbo Polygala Bioengineering Company, and Taq enzyme was purchased from Sanbo Polygala Bioengineering Company.
[0081] A total of 4 tubes of PCR system were configured, numbered from 2 to 5.
[0082...
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