JRD218PCR experiment

polymerase chain reaction, PCR is from the date of birth It determines that it is not only a qualitative method of detecting nucleic acid molecules, but also a powerful tool that can be quantified for nucleic acid molecules. With the continuous advancement of molecular biology research, quantitative PCR technology has achieved long-term development, and real-time quantitative PCR (Real-TimeQuantitative PCR) technology is a meaningful quantitative PCR technology. The so-called real-time quantitative PCR refers to the initial amount of the specific product to instantly monitor the strongly monitoring of the strong monitoring of fluorescent signals during the PCR index amplification, and inform the initial amount of the object gene.

Taqman fluorescence probe PCR amplification When adding a pair of primers, add a specific fluorescent probe, which The probe is a oligonucleotide, and a reporting fluorescent group and a quenched fluorescent group are respectively labeled. When the probe is complete, the fluorescent signal emitted by the report group is quenched by the group; when the PCR amplification, the 5 u0026 # 39; -3 u0026 # 39; the external transferase activity will degrade the probe enzyme, so that the report fluoresce The group and the quenching fluorescence group are separated, so that the fluorescence monitoring system can receive a fluorescent signal, that is, each of the a DNA chain is amplified, there is a fluorescent molecule formed, and the accumulation of the fluorescent signal is synchronized with the PCR product.

Sybr Green fluorescent dye
Sybr Green is a DNA binding dye that is non-specially incorporated into the double-stranded DNA. In the free state, it does not emit fluorescence, but once it is incorporated into the double-stranded DNA, fluorescence can be emitted. Its great advantage is that it can be mounted with primers, templates for the reaction system. From the economic point of view, it is much cheaper than other probes. However, since it can be combined with the double-stranded DNA, it will affect the reliability and repetition of quantitative results once there is a non-specific amplification in the reaction system. To avoid this unfavorable factor, you can eliminate non-specific effects by selecting good primers and optimizing the reaction conditions.



The steps are as follows: 1. Designed and synthesized a RealTime PCR primer. 2. Optimization of PCR reactions containing SG (Sybr u0026 Reg; Green) using Promega TAQDNA polymerase after primer dissolution. 3. Customer sample RNA extraction Experimental objects for tissue samples, take appropriate amount (50-100 mg) fresh tissue sample or orthimate tissue sample plus 1 mL of RNA extract reagent Trizol (Invitrogen), and homogenate after extracting RNA.
The experimental object is a cell sample, each sample takes 1 u0026 Times; 106 ~ 1 u0026 Times; 107 cells, PBS cleaning cells, PBS plus 1 mL of RNA extract reagent Trizol (Invitrogen), cleaves RNA [123 ] 4. RNA quality detection
a. Determination of the absorption value at the spectrophotometer 260 nm and 280 nm to calculate its concentration and evaluate RNA purity
b. Using Ambion The formaldehyde electrophoresis reagent performs denaturing agarose gel electrophoresis, detects RNA purity and integrity.
5. Use reverse transcriptase (Promega) to react to cDNA reverse the sample RNA.
6. Preparation of standard curve samples:
For relative quantification, the purpose of the samples of the sample and the manager gene are first to be amplified, and the product is diluted to perform a standard curve.
7. Standard curve samples and samples to be tested were added to the SG-containing RealTime PCR reaction solution (where TaqBeadTM thermal start-up polymerase came from Promega), RealTime PCR amplification and detection.
8. Detection results for standard curve analysis: quantify the target gene to be tested. The relative quantitative experiments need to further use the target gene measurement value of the sample to be measured at the manager genetic value to correct the error, and the resulting result represents the relative content of the purpose gene of a sample.
9. Provide experimental report: including detailed experimental methods and real-time fluorescent quantitative results related chart