HCY™ Taq DNA Polymerase Kit

The following is the general usage method of the HCY™ Taq DNA Polymerase Kit:

1. Preparation work:
   • Reagent preparation: Take out the HCY™ Taq DNA Polymerase Kit from the refrigerator to ensure that all components in the kit are complete and not expired. Usually, the kit will include Taq DNA polymerase, buffer (containing magnesium ions and other components), dNTPs (deoxyribonucleoside triphosphates), and an appropriate amount of sterile water, etc. Before use, let the reagents stand at room temperature for a period of time to make their temperature reach room temperature to avoid affecting the experiment due to temperature differences.
   • Experimental equipment preparation: Prepare PCR tubes or plates, pipettes, centrifuges, PCR instruments and other experimental equipment, and ensure that these equipment are clean and free from contamination.
   • Template DNA preparation: According to the purpose of the experiment, prepare an appropriate amount of template DNA. The template DNA can be extracted genomic DNA, plasmid DNA, etc., and its quality and concentration are crucial to the success of the PCR reaction. Use methods such as spectrophotometry to detect the concentration and purity of the template DNA to ensure that it meets the experimental requirements. Generally, the concentration of the template DNA is more suitable in the range of 10 – 100 ng/μL, but the specific concentration still needs to be optimized according to the experimental situation.
2. Prepare the PCR reaction system:
   • Determine the total volume of the reaction system: Determine the total volume of the PCR reaction system according to the experimental needs and the specifications of the PCR tube or plate. Generally, the common total volume of the PCR reaction system is 20 – 50 μL.
   • Add buffer: Take an appropriate amount of the buffer provided in the kit and add it to the PCR reaction system. The function of the buffer is to provide a suitable pH environment and ion concentration for the PCR reaction to ensure the activity of the Taq DNA polymerase. Usually, the addition amount of the buffer is about 1/10 of the total volume of the reaction system, but the specific dosage still needs to refer to the kit instructions.
   • Add dNTPs: dNTPs are the raw materials for synthesizing new DNA chains in the PCR reaction, including dATP, dGTP, dCTP, and dTTP. Add an appropriate amount of the dNTPs mixture to the reaction system to make its final concentration reach 200 – 250 μM each. For example, if the total volume of the reaction system is 25 μL, the addition amount of the dNTPs mixture can be 2 μL (the concentration of dNTPs provided in the kit is usually relatively high, and it needs to be diluted and added according to the actual situation).
   • Add primers: Primers are short-chain DNA fragments that specifically bind to the template DNA and are used to guide the Taq DNA polymerase to synthesize new DNA chains. According to the experimental design, add an appropriate amount of forward and reverse primers to make the final concentration of the primers 0.1 – 1.0 μM. The design and selection of primers are very important, and it is necessary to ensure their specificity, length, GC content, etc. meet the experimental requirements. Primer design software can be used for primer design, and the validity of the primers can be verified through experiments.
   • Add template DNA: Add an appropriate amount of template DNA to the reaction system. The addition amount of the template DNA should be determined according to its concentration and experimental needs, and it is generally recommended that the amount of the template DNA is between 1 and 100 ng. If the amount of the template DNA is too much, non-specific amplification may occur; if the amount of the template DNA is too little, sufficient PCR products may not be obtained.
   • Add Taq DNA polymerase: Finally, add an appropriate amount of HCY™ Taq DNA polymerase. The addition amount of the Taq DNA polymerase is usually 0.5 – 2.5 U/50 μL reaction system, and the specific dosage needs to be optimized according to the kit instructions and the experimental situation. Note that when adding the enzyme, avoid the enzyme solution from contacting the tube wall to prevent the enzyme solution from adhering to the tube wall and affecting the accuracy of the reaction system.
   • Add water to the total volume: Use sterile water to fill the reaction system to the predetermined total volume.
3. PCR reaction condition settings:
   • Pre-denaturation: Put the PCR tube or plate containing the PCR reaction system into the PCR instrument, and set the pre-denaturation temperature to 94 – 98℃, and the pre-denaturation time to 2 – 5 minutes. The purpose of pre-denaturation is to completely denature the template DNA and unwind the double strands so that the primers can bind to the template DNA.
   • Denaturation: Set the denaturation temperature to 94 – 98℃, and the denaturation time to 15 – 30 seconds. The denaturation step is to keep the template DNA in a single-stranded state in each PCR cycle so that the primers can bind to the template DNA.
   • Annealing: The annealing temperature is determined according to the melting temperature (Tm value) of the primers, which is generally about 5℃ lower than the Tm value. The annealing time is usually 15 – 30 seconds. The annealing step is to make the primers specifically bind to the template DNA.
   • Extension: Set the extension temperature to about 72℃ (the optimum extension temperature of the Taq DNA polymerase), and the extension time is determined according to the length of the amplified fragment, generally, for each 1 kb fragment amplified, it takes 1 – 2 minutes of extension time.
   • Cycle number: Determine the cycle number of the PCR reaction according to the experimental needs, generally 25 – 40 cycles. Excessive cycle numbers may lead to an increase in non-specific amplification; too few cycle numbers may not obtain sufficient PCR products.
   • Final extension: After completing the set cycle number, set the final extension temperature to 72℃, and the final extension time to 5 – 10 minutes. The purpose of the final extension is to ensure that the products that have not been completely extended in the PCR reaction can be completely extended to improve the yield and quality of the PCR products.
4. PCR product detection:
   • Electrophoresis detection: After the PCR reaction is completed, take out the PCR tube or plate, take an appropriate amount of the PCR product and mix it with the loading buffer, and then perform agarose gel electrophoresis detection. Add an appropriate amount of nucleic acid dye, such as ethidium bromide (EB) or SYBR Green, etc., to the agarose gel, add the mixed PCR product sample to the loading hole of the gel, and perform electrophoresis under a certain voltage. After electrophoresis, observe the bands of the PCR products under ultraviolet light to determine whether the PCR reaction is successful.
   • Other detection methods: In addition to electrophoresis detection, other detection methods can also be selected according to the experimental needs, such as real-time fluorescence quantitative PCR detection, sequencing, etc.

It should be noted that the above usage method is only a general guide, and the specific usage method may vary depending on the kit. Before using the HCY™ Taq DNA Polymerase Kit, be sure to read the kit instructions carefully and operate according to the experimental requirements. At the same time, in order to ensure the accuracy and reliability of the experimental results, it is recommended to conduct negative control and positive control experiments.