Views:0 Author:Site Editor Publish Time: 2021-11-20 Origin:Site
When PCR machines detect micro-infection factors, they are prone to various problems due to contamination. Therefore, when operating PCR machines, operators should strictly follow some operating procedures to minimize possible PCR contamination or eliminate the occurrence of contamination and other problems. The following will describe the use of PCR machines and precautions.
Here is the content list:
l Instructions for the operation of the PCR machine.
l Notes on the use of PCR machines.
l PCR instrument care and maintenance methods.
1. Primers: determine the specificity of PCR machine amplification. Different primers are used depending on the DNA testing, and each pathogenic microorganism has its own specific primers.
2. Heat-resistant DNA polymerase: This enzyme is isolated from heat-resistant bacteria and can withstand high temperatures of 90°C-100°C.
3. 10×PCR machine buffer: 500mmol/l KCl; 100mmol/L Tris-HCl (pH8.4,20℃) 150mmol/L MgCl2,1mg/ml gelatin. 10×PCR buffer was purchased together with the enzyme.
4.5 mmol/L dNTP PCR machine stock solution: Combine 100 mg each of dATP, dCTP, dGTP, and dTTP sodium salts, add sterilized deionized water to dissolve, adjust pH to neutral with NaOH, dispense 300 μl each and store at -20℃. dNTP concentration is determined by the UV absorption method. The dNTP solutions available now are commercialized products.
5. DNA template: The specimen to be tested is treated with a treatment solution. Different treatments and operations vary, but the purpose is to expose the DNA being tested in the PCR machine specimen.
1. The PCR machine should be placed on a horizontal and sturdy platform with a matching voltage of the external power system and a good grounding wire is required.
2. The ambient temperature is kept at about 23℃ and the humidity is kept at about 60%.
3. PCR machine should be equipped with a power ≥ 3000W voltage regulator.
4. It should be cleaned and maintained regularly.
5. When using the PCR machine, the above usage steps should be strictly observed.
1. Cleaning of the cuvette first open the lid of the PCR machine, then soak the cuvette with 95% ethanol or 10% cleaning solution for 5 min, then clean the contaminated wells; aspirate the liquid with a micropipette and use a cotton swab to absorb the remaining liquid; turn on the PCR machine, set the PCR program to maintain the temperature at 50°C and make it run, and let the residual liquid evaporate and remove. Usually, 5-10 min is sufficient.
2. Hot cover cleaning for fluorescent quantitative PCR machine, when there is fluorescent contamination, and this contamination is not from the cuvette, or when there is contamination or residue affect the PCR machine hot cover of the loose, you need to use compressed air or pure water to clean the bottom of the pad cover, to ensure that the cuvette hole is clean, no dirt blocking the optical path.
3. Cleaning the external surface of the PCR instrument cleaning the external surface of the instrument can remove dust and grease, but not up to the effect of disinfection. Choose a non-corrosive cleaning agent to clean the external surface of the PCR machine.
4. Replace the fuse first turn off the PCR machine, unplug it, open the fuse box next to the power jack, replace it with a spare fuse, and observe whether it returns to normal.
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