Reverse transcription-polymerase chain reaction (RT-PCR)
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) can: (1) analyze gene transcripts; (2) obtain target genes; (3) synthesize cDNA probes; (4) construct RNA Efficient transcription system.
The principle of the experimental method The principle of Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is to extract the total RNA in tissues or cells, use the mRNA as a template, and use Oligo (dT) or random primers Reverse transcription into cDNA using reverse transcriptase. Then use cDNA as a template for PCR amplification to obtain the target gene or detect gene expression.
Experimental materials: tissue cell reagents, kits: RNA extraction reagents dNTP mixture Taq DNA polymerase first strand cDNA synthesis kit instruments, consumables: centrifuge tube centrifuge Water bath PCR tube electrophoresis instrument gel image analysis system pipette pipetting Gun centrifuge tube box
Experimental procedure
1. Extraction of total RNA
2. Synthesis of cDNA first strand At present, there are a variety of cDNA first strand kits sold by reagent companies. The principle is basically the same, but the operation steps are different. Take the SuperScriptTM Preamplification System for First Strand cDNA Synthesis kit provided by GIBICOL as an example. 1. In a 0.5 ml microcentrifuge tube, add 1-5 μg of total RNA and add an appropriate amount of DEPC H2O to make the total volume up to 11 μl. Add 10 μM Oligo (dT) 12-18 1 μl to the tube, gently mix and centrifuge; heat at 2.70 ° C for 10min, immediately insert the microcentrifuge tube into the ice bath for at least 1min; 3. Take 0.5 ml PCR tube and add in sequence The following reagents: first strand cDNA 2 μl; upstream primer (10 pM) 2 μl; downstream primer (10 pM) 2 μl; dNTP (2mM) 4 μl; 10 × PCR buffer 5 μl; Taq enzyme (2 u / μl) 1 μl. Mix gently and centrifuge. Incubate at 42 ° C for 2-5 min; 4. Add 1 μl of Superscript II and incubate in a 42 ° C water bath for 50 min; 5. Heat at 70 ° C for 15 min to stop the reaction; 6. Insert the tube into ice and add RNase H 1 μl, 37 Incubate at ℃ for 20 min to degrade residual RNA. Store at -20 ℃ for future use.
3. PCR 1. Take a 0.5 ml PCR tube and add the following reagents in sequence: 2 μl of the first strand cDNA; 2 μl of the upstream primer (10 pM); 2 μl of the downstream primer (10 pM); 4 μl of dNTP (2 mM); 10 × PCR buffer 5 μl; Taq enzyme (2 u / μl) 1 μl; 2. Add appropriate amount of ddH2O to make the total volume up to 50 μl. Mix gently and centrifuge; 3. Set up PCR program. Amplify 28-32 cycles under appropriate temperature parameters. In order to ensure the reliability and accuracy of the experimental results, a pair of specific primers for internal control (such as G3PD) can be added during PCR amplification of the target gene, and the internal control DNA can be amplified at the same time as a control; Electrophoresis, observe the results under ultraviolet light; 5. Density scanning, result analysis: A gel image analysis system is used to perform density scanning on the electrophoretic strips.
4. 1. Selection of reverse transcriptase (1) Money murine leukemia virus (MMLV) reverse transcriptase: has strong polymerase activity, and RNase H activity is relatively weak. The optimum temperature is 37 ℃; (2) Avian myeloblastoma virus (AMV) reverse transcriptase: it has strong polymerase activity and RNase H activity. The optimum temperature is 42 ℃; (3) Thermostable reverse transcriptases of thermophilic microorganisms such as Thermus thermophilus and Thermus flavus: In the presence of Mn2 +, allow high temperature reverse transcription of RNA to eliminate the secondary structure of RNA template (4) RNase H-mutant of MMLV reverse transcriptase: trade names Superscript and SuperScript II. This enzyme can convert a larger portion of RNA into cDNA than other enzymes. This feature allows the synthesis of longer cDNA from mRNA templates that contain secondary structures and are difficult to reverse transcribe at low temperatures.
2. Selection of synthetic cDNA primers (1) Random hexamer primers: When a specific mRNA contains a sequence that terminates reverse transcriptase and it is difficult to copy its full-length sequence, the non-specific random hexamer primer can be used Primers to copy full-length mRNA. With this method, all RNA molecules in the system all serve as the first strand cDNA template, and PCR primers confer the required specificity during the amplification process. Usually 96% of the cDNA synthesized with this primer is derived from rRNA. (2) Oligo (dT): is a method specific to mRNA. Since most eukaryotic cell mRNAs have a Poly (A +) tail at the 3 'end, this primer is paired with it, and only the mRNA can be transcribed. Since Poly (A +) RNA only accounts for 1-4% of the total RNA, the cDNA synthesized by this kind of primer is smaller than the random hexamer as a primer and the resulting cDNA is smaller in number and complexity. (3) Specific primers: The most specific priming method is to use oligonucleotides containing the complementary sequence of the target RNA as primers. If two specific primers are used for the PCR reaction, the synthesis of the first strand can be combined with the 3 'end of the mRNA The closest paired primer starts. The use of such primers produces only the required cDNA, leading to more specific PCR amplification.
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