polymerase chain reaction (PCR)
polymerase chain reaction (PCR)
The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
polymerase chain reaction (PCR)
Materials and Instruments
Heat-stable DNA polymerase Parasite DNA Forward primer Reverse primer Template DNA Move I. Materials For more product details, please visit Aladdin Scientific website.
Amplification buffer Chloroform dNTP storage solution
Polyacrylamide gel or agarose gel Shielded tip Tubes Forward-displacement pipettes PCR instruments
1. Buffers and solutions
10X Amplification Buffer
Chloroform
4 dNTP storage solution (20 mmol/L, pH 8.0)
2. Enzyme and buffer
Heat-stabilized DNA polymerase
3. gels
Polyacrylamide gel or agarose gel
4. nucleotides and oligonucleotides
Bystander DNA
Forward primer (20 μmol/L) and reverse primer (20 μmol/L) Dissolve in water 
Template DNA
5. Special equipment
Shielded tips for automated micropipettes
Centrifuge tubes (0.5 ml, thin-walled for amplification reactions)
Positive displacement pipettes
6. PCR instrument
II. Methods
1. Add the components to the wells of a 0.5 ml sterilized centrifuge tube, amplification tube or sterilized titration plate in the following order:
10X Amplification Buffer 5 μl
20 mmol/L 4 dNTP mix (pH 8.0) 1 μl
20 μmol/L forward primer 2.5 μl
20 μmol/L reverse primer 2.5 μl
1~5 U/μl Heat-stabilized DNA polymerase 1~2 units
H2O 28~33 μl
Template DNA 5~10 μl
Total volume 50 μl 
2. If the PCR instrument is not equipped with a heated lid, a drop of light mineral oil (approx. 50 μl) should be added to the top of the reaction mixture to prevent the sample from evaporating during multiple cycles of the PCR reaction. If a hot start protocol is applied, a layer of paraffin oil may be added to the top of the reaction mixture. Place a microcentrifuge tube or microtiter plate on the PCR instrument.
3. Perform PCR amplification as described below. Typical procedures are denaturation, denaturation, and polymerization (extension reaction); the corresponding cycling conditions with temperatures are listed below: 
These cycle numbers are set for a 50 μl reaction system in a 0.5 ml thin-walled centrifuge tube, which is incubated for target gene amplification on a PCR instrument, either a Perkin-Elmer 9600 or 9700; an Eppendorf Master cycler PCR instrument; or an MJ Research PTC100. The cycle numbers and temperature settings here may also be appropriate for other types of equipment and different reaction volumes.
The polymerization reaction should be set up to polymerize at a rate of 1000 bp per minute based on the length of the target gene. The final procedure for most PCR instrument setups is to incubate the amplified samples at 4°C until the amplified samples are removed from the PCR instrument. Some amplified samples can be left on the PCR instrument overnight and stored at -20°C later.
4. 5-10 μl of each amplified sample is withdrawn and analyzed by agar gel electrophoresis or polyacrylamide gel electrophoresis, and a DNA marker is used to determine the size of the amplified fragments. The gel is usually stained with EB (ethidium bromide) or SYBR gold particles to observe the amount of amplification and fragment size later.
A successful amplification reaction should produce DNA fragments of the size we expect. Amplified bands can be identified by DNA sequence analysis, Southern hybridization and restriction endonucleic acid digestion profiles.
If these identifications indicate that the amplification product is correct, the different lanes of the gel electrophoresis can be further analyzed; the efficiency of the PCR amplification can be determined from the brightness and thickness of the target bands of the corresponding molecular weights of the two tubes of the positive control samples; the negative control samples should not have any corresponding bands near the target bands.
5. If mineral oil is used to coat the top layer of sample liquid in the microcentrifuge tube, it can be removed by 150 μl of chloroform extraction at the end of the reaction.
Inside the PCR reaction tube, the interface between the aqueous phase containing the amplified DNA fragments and the upper layer of mineral oil forms a crescent, and the aqueous phase underneath the crescent also contains microgels. The aqueous phase can be carefully pipetted into a new centrifuge tube. This mineral oil must be removed from the sample for many purposes (e.g., purification of amplified DNA samples using a Cemricon Microconcentrator or further cloning of such amplification products).
PCR Optimization
At the beginning of PCR optimization, the amplification results of PCR methods using new DNA templates, primers or heat-stable DNA polymerase are generally not optimal. Such PCR reactions are often required to minimize nonspecific amplification and/or increase the yield of the target DNA product. 
