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1. The basic elements of PCR and the principle of amplification
The basic elements are the same as those of DNA replication. The DNA to be copied is called the template, it can be double-stranded DNA or single-stranded DNA, and the final amplified product is in double-stranded state. Primers are the pioneers of DNA replication, just like the crystal nuclei in the crystallization process, which guide the synthesis of DNA. In PCR amplification, synthetic oligonucleotides are generally used as primers. DNA polymerase is the driving force for DNA replication. When dNTP and other substrates are present, the complementary DNA strand is synthesized along the template DNA under the guidance of primers.
2. The steps of PCR amplification
First put the template DNA at 92 ℃ -96 ℃, denaturation (daturation), so that dsDNA melts at high temperature into ssDNA, and thermal denaturation does not change its chemical properties; then annealing (annealing), the temperature is reduced to 37 -72 ℃, so that the primer and the complementary region of the template are combined; Finally, under 72 ℃, DNA polymerase will continuously add dNTP to the 3 "-OH end of the primer to synthesize DNA, this step is called extension (extension). The repetition of these three thermal reaction processes is called a cycle. After 20-40 cycles, a large number of DNA fragments between the two primer sequences can be amplified. Figure 1-1 shows the first four rounds of the PCR reaction.
Figure 1-1: Schematic diagram of the first four rounds of PCR reaction.
Principles of PCR amplification product identification and purification experiment
Agarose gel electrophoresis was used to identify PCR amplification products. (The principle refers to agarose gel electrophoresis detection DNA experiment)
In this experiment, TaKaRa's Agarose Gel DNA Purification Kit was used. This kit is a kit for recovering and purifying DNA fragments from agarose gel. The kit uses a unique gel melting system, combined with DNA preparation membrane technology, has the characteristics of high efficiency, fast and convenient, a full set of operations can be completed in 30 minutes. With this kit, up to 10 μg of DNA fragments (100 bp to 30 kb) can be purified each time, and the recovery rate is as high as 50 to 80%. The DNA fragments recovered and purified by this kit have high purity and good integrity, and can be directly used in various molecular biology experiments such as ligation reaction, PCR amplification, DNA sequencing and the like.
The DNA fragments recovered by the gel purification kit are stored at a low temperature of -20 ° C to delay the degradation of DNA.
Experimental reagent
1. PCR amplification products
2. Agarose Gel DNA Purification Kit (TaKaRa)
3. Agarose
4. 1 × TAE
5. 6 × Loading Buffer
6. DNA Marker 2000
Laboratory equipment
1. Agarose gel electrophoresis system
2. Ultraviolet transmission instrument
3. Desktop centrifuge
4. Pipette (matching pipette tip)
5. -20 ℃ low temperature refrigerator
6. Analytical balance
Experimental Materials
Single-sided blade
2. Purification kit
3. 1.5mL centrifuge tube
4. Ultrapure water (sterile)
Experimental procedure
1. Make an agarose gel using 1 × TAE buffer, and then perform agarose gel electrophoresis on the target DNA. (Refer to agarose gel electrophoresis detection DNA experiment)
2. Cut out the agarose gel containing the target DNA (approximately 600bp) under the UV lamp, and use the paper towel to suck up the liquid on the surface of the gel. At this time, care should be taken to remove the gel that does not contain the DNA portion of interest, try to reduce the volume of the gel, and improve the DNA recovery rate.
Note: When cutting the gel, be careful not to expose the DNA to UV light for a long time to prevent DNA damage.
3. Shred the rubber block. After the gel block is minced, the melting time of the gel block in step 6 can be accelerated, and the DNA recovery rate can be improved.
4. Weigh the rubber block and calculate the rubber block volume. When calculating the volume of the rubber block, 1mg = 1μL is used for calculation.
5. Add the DR-IBuffer to the rubber block. The amount of DR-I Buffer is as follows:
Use of DR-I Buffer
1% 3 gel volume
1% -1.5% 4 gel volume
1.5% -2% 5 gel volume
6. After evenly mixing, heat at 75 ℃ to melt the gel (low melting agarose gel only needs to be heated at 45 ℃) At this time, the mixture should be shaken intermittently to melt the rubber block (about 6 to 10 minutes).
Note: The gel block must be fully melted, otherwise it will seriously affect the DNA recovery rate.
7. Add 1/2 volume of DR-II Buffer to the melt of the above gel and mix evenly. When isolating DNA fragments smaller than 400 bp, isopropyl alcohol with a final concentration of 20% should be added to this solution.
8. Place the Spin Column in the kit on the CollectionTube.
9. Transfer the solution from operation 7 above to SpinColumn, centrifuge at 12000 rpm for 1 min, and discard the filtrate.
Note: If the filtrate is centrifuged once again in SpinColumn, the DNA recovery rate can be improved.
10. Add 500 μL of Rinse A to the Spin Column, centrifuge at 12000 rpm for 30s, and discard the filtrate.
11. Add 700 μL of Rinse B to the Spin Column, centrifuge at 12000 rpm for 30s, and discard the filtrate.
12. Repeat step 11.
13. Place the Spin Column on a new 1.5 mL centrifuge tube, add 25 μL of sterile distilled water or Elution Buffer to the center of the SpinColumn membrane, and let stand at room temperature for 1 min.
Note: The use of sterilized distilled water or ElutionBuffer heated to 60 ° C is beneficial to improve the elution efficiency.
14. Centrifuge at 12000 rpm for 1 min to elute DNA.
Store the purified DNA fragments at a low temperature of -20 ℃.
The above is the basic principle of PCR compiled by the editor and the identification and purification of amplified products
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