Author: Li Aili, etc. Source: Biotechnology Bulletin
Typical PCR operations are only listed here for the general PCR procedure. The optimization of the specific influencing factors will be discussed in the second section of this chapter. The necessary optimizations are required before each specific operation.
(1) Reagents (1) Primers are different depending on the DNA to be amplified. The design of the primers is shown in Section III of this chapter.
(2) Thermostable DNA polymerase: This enzyme is isolated from heat-resistant bacteria and can withstand high temperatures (93-100c).
The characteristics of different heat-resistant DNA polymerases are detailed in Section 4 of this chapter. Perkin-E1mer-cetus,
N, F, Bio1ab, PharMacia, domestic and American companies, Fudan. The University and the Chinese Academy of Medical Sciences Friendship Company have supplies.
(3) 10x PCR buffer: 500mm01/L KCl; 100mmol/L Tris-HCl (pH8.4, 20c),
150mmol/L MgCl2, lmg/m1 gelatin o “â€
(4) 5mmo1/L dNTP stock solution: combine 100mg of dATP, dCTP, dGTPT and dTTP sodium salt, add 3m1 sterilized deionized water to dissolve, adjust pH to neutral with NaoH, and pack 300v1 each, (-) Store at 20 ° C
The dNTP concentration is preferably determined accurately by the uv absorption method. Also. There is now a supply of neutral dNTP solution (Sigma and Pharmacia, etc.).
(5) Specimen processing reagents: different reagents required for different specimens, depending on the specific conditions [see section 6 of this chapter) G
(2) The operating procedure using PcR amplification is basically the same, except that the different reaction systems and cycle parameters are selected according to the difference between the primer and the target sequence (see section::): The basic operation is to make the PcR essential reaction component. It is added to a microcentrifuge tube and then subjected to cyclic amplification under certain cycling parameters. Each laboratory or everyone has different operating habits, but must follow certain operating practices. We recommend the following procedure as this will maximize the success rate of the reaction.
(1) Add sequentially to a microcentrifuge tube:
ddH2 O to the final volume (final volume 50~100ul)
10 x PCR buffer 1/10 volume
dNTP each 200umol/L
Primers 1umol/L each
The DNA template 10*10-10*10*10*10*10 copies were mixed and centrifuged for 15 seconds to collect the reaction components at the bottom of the tube.
(2) Add paraffin oil 50-100 ul to the surface of the reaction liquid to prevent evaporation. The reaction tube was denatured at 97c for 7 min (chromosome
DNA) or 5min (plasmid DNA).
(3) When cooling to the extension temperature, add 1-5 U Taq DNA polymerase at this temperature for 1 min.
(4) Denaturation of the template DNA at a denaturation temperature for a suitable period of time.
(5) The primer is hybridized to the template for a certain time at the renaturation temperature.
(6) Extending the renature primer at the extension temperature for a suitable period of time.
(7) Repeat (4) one (6) steps 25-30 times. Each time is a PcR cycle o
(8) Amplification of the amplified product by micro-agar gel electrophoresis (see section 7 of this chapter) o
The above process can be carried out by PcR automatic thermal cycler, or it can be set by 3 constant temperature water baths.
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