Introduction to PCR Lab
The Pcr laboratory is also called the gene amplification laboratory, the DNA laboratory, and the genetic testing laboratory. PCR is an abbreviation for Polymerase Chain Reaction. It is a molecular biology technique used to amplify specific DNA fragments and can be seen as a special DNA replication outside the organism. Through the DNA gene tracking system, the virus content in the patient can be quickly grasped, and the accuracy is up to the nanometer level. The number of hepatitis B virus present in the patient is accurately detected, whether it is copied, whether it is contagious, how infectious is necessary, whether it is necessary to take medicine, liver Whether the function has abnormal changes can timely determine which type of antiviral drugs are most suitable for the patient, determine the efficacy of the drug, and provide a reliable test basis for clinical treatment.
Function: It can scientifically, accurately and real-time control the patient's condition, combined with anti-HBV and T cell immunity to break the immune tolerance, block the hepatic virus replication therapy, effectively decompose the hepatitis virus, and solve the HBV virus variability. Drug resistance, virus replication template is difficult to treat, human immune tolerance is not easy to break and other medical problems, can quickly eliminate clinical symptoms, and effectively inhibit hepatitis B virus replication, significantly accelerate the e-antigen, antibody seroconversion rate, kill blood and liver cells The virus inside provides long-term protection against reinfection, effectively blocking and reversing the process of liver fibrosis and cirrhosis.
construction plan
1. Establish a sample preparation area
This area is dedicated to the preparation of samples, and precautions should be taken when preparing and handling reagents for nucleic acid extraction: (1) PCR products and DNA clones with sequences to be amplified cannot be manipulated in this room. (2) Tissue culture, tissue samples and serum samples are taken into the sample preparation process to extract DNA or RNA according to the needs of the application. (3) Tools for sample processing cannot be used as a tool for ordinary molecular cloning, nor can it be used as an operation target sequence. (4) DNA samples should be handled with a special protective or positive pressure piston Pipette to prevent aerosol build-up when the sample is aspirated. (5) Large volume samples should be pipetted with a sterile disposable pipette. (6) The tube should be briefly centrifuged before opening to reduce the generation of aerosol, and the tube cannot be broken by force, which will generate aerosol. (7) You should wear lab coats and gloves at all times. Gloves should be replaced frequently, especially during each step of the extraction process. The lab coat should be used exclusively for sample preparation and often cleaned.
2. Additional steps in sample preparation and RNA-PCR RNA-PCR require additional sample manipulation, which increases the chance of contamination between samples. To avoid this problem, a reverse transcription step can be performed in the sample preparation area. Methods for applying UNG to prevent contamination in RNA-PCR have also been reported.
3. Establish a pre-PCR region.
This is specifically designed to prepare for various reactions, this area must be kept clean and there is no contamination from cloning and sample preparation. The pre-PCR zone must have reagents and preparations, especially positive pressure piston pipettes dedicated to the pre-PCR zone.
4. Operation of PCR laboratory reagents.
(1) All solutions used should be free of nucleic acid and/or nuclease (DNase and RNase) contamination. (2) The water used in all PCR reagents should be of high quality - freshly distilled deionized water, filtered with 0.22 μm, and autoclaved. (3) It is recommended to add an antimicrobial agent such as sodium azide to the reagent stored at 20 ° C to 25 ° C. Adding 0.025% sodium azide to the amplification reagent or sample preparation reagent does not inhibit the amplification reaction. (4) The reagents used should be prepared in a large volume. Experiment to see if the reagents are satisfactory, and then store them in an amount that is only enough for one use. (5) Disposable sterile bottles and tubes should be used during all reagent and sample preparation. (6) Newly prepared reagents should be tested before being used to prepare new specimens. (7) Sample preparation and pipettes used in the pre-PCR area should be carefully stored when not in use.
5. Establish a PCR mixture in the pre-PCR region.
(1) The ready-to-use "master mix" solution can be dispensed, dispensed, and stored at -20 ° C or 4 ° C, which is useful when the laboratory only involves amplification of one or a few specific sequences. (2) If your laboratory uses multiple sets of primers, so that it is not economical to formulate a single reaction mixture that includes all reagents, consider dispensing the PCR components for one day. (3) As a rule, a set of negative, weakly positive, and strong positive control samples should be saved to analyze the efficiency and cleanliness of the sample preparation and pre-PCR process. Also, you want to verify your sample buffer by using a known weak positive sample to prove that it does not contain amplification inhibitors. (4) Negative samples should be prepared simultaneously with each set of samples to analyze whether there is contamination between the sample and the sample and whether there is contamination of the PCR product. The negative control should include all reagents other than nucleic acids. (5) When used as a positive control, there are two reasons for determining the minimum number of nucleic acids that should be used. (6) Since a control reaction is necessary, the characteristics of the control template should be considered.
6. Methods to control pollution.
Energetic methods have been devised to eliminate one form of contamination - using UNG, a technique that effectively eliminates contamination caused by PCR products. Another way to control pollution is to use ultraviolet light. This method does not completely eliminate the pollution problem, but can reduce the pollution by several orders of magnitude.
7. After the PCR in the post-PCR region is completed, the sample needs to be analyzed and the data interpreted. A special place for the post-reaction sample should be set aside. The reagents, disposable consumables and instruments used in post-PCR activities must be used exclusively for this purpose. Never use reagents or instruments from this area of the laboratory for any pre-PCR activities.
Application place
Third-party medical laboratory, judicial appraisal agency, clinical medical testing institution, criminal investigation technology appraisal agency, agricultural and animal husbandry genetic testing, food industry genetically modified food testing, food and drug supervision, disease control center, etc.
PCR Laboratory Equipment list
(1) Reagent storage and preparation area
1, 2-8C and -15C refrigerator
2, mixing device
3, micro sampler (covering 1-1000ul)
4, mobile UV lamp (near work surface)
5, consumables: disposable gloves, disposable absorbent paper, high pressure resistant Centrifuge Tube and sampler tip (with filter)
6, special overalls and work shoes
7, special office supplies
(2) Specimen preparation area
1, 2-8C refrigerator, -20C or -80C refrigerator
2, high-speed desktop refrigerated centrifuge
3, the mixer
4, water bath or heating module
5, micro sampler (covering 1-1000ul)
6, movable UV lamp (near work surface)
7, ultra-clean workbench
8, consumables: disposable gloves, disposable absorbent paper, high pressure treated centrifuge tube and sampler tip (with filter)
9, special overalls and work shoes
10, special office supplies
If you need to deal with macromolecular DNA, you should have an ultrasonic water bath.
(3) Amplification reaction mixture preparation and amplification zone
1. Nucleic acid amplification instrument
2, micro sampler (covering 1-1000ul)
3, movable UV lamp (near work surface)
4, consumables: disposable gloves, disposable absorbent paper, high pressure resistant centrifuge tube and sampler tip (with filter)
5, special overalls and work shoes
6, special office supplies
(4) Amplification product analysis area
Depending on the method of inspection. The basic equipment is as follows:
1, micro sampler (covering 1-1000ul)
2, movable UV lamp (near work surface)
3, consumables: disposable gloves, disposable absorbent paper, sampler tips (with filter)
4, special overalls and work shoes
5, special office supplies
