Principle and experimental process of PCR amplification product identification and purification
Experimental principle
The PCR amplification product was identified by agarose gel electrophoresis. (Principle reference to agarose gel electrophoresis for DNA testing)
This experiment used TaKaRa's Agarose Gel DNA Purification Kit, which is a kit for recovering and purifying DNA fragments from an agarose gel. The kit uses a unique gel melting system combined with DNA preparation membrane technology, which is efficient, fast and convenient. The complete operation takes only 30 minutes. Up to 10 μg of DNA fragment (100 bp to 30 kb) can be purified each time using the kit, and the recovery rate is as high as 50 to 80%. The purified and purified DNA fragment of the kit has high purity and good integrity, and can be directly used in various molecular biological experiments such as ligation reaction, PCR amplification and DNA sequencing.
The DNA fragment recovered by the gel purification kit is stored at a low temperature of -20 ° C to delay the degradation of DNA.
Experimental reagent
PCR amplification product
2. Agarose Gel DNA Purification Kit (TaKaRa)
3. Agarose
4. 1×TAE
5. 6×Loading Buffer
6. DNA Marker 2000
experiment apparatus
Agarose gel electrophoresis system
2. UV Transmitter
3. Benchtop centrifuge
4. Pipette (matching gun head)
5. -20 ° C 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. Agarose gel was prepared using 1×TAE buffer, and then the target DNA was subjected to agarose gel electrophoresis. (Refer to agarose gel electrophoresis for DNA assay)
2. Cut the agarose gel containing the DNA of interest (about 600 bp) under a UV lamp and blot the liquid on the surface of the gel with a paper towel. At this point, care should be taken to remove the gel that does not contain the DNA of interest, try to reduce the gel volume and increase the DNA recovery rate.
Note: Please be careful not to expose the DNA to UV light for a long time to prevent DNA damage.
3. Cut the pieces of glue. After the rubber block is chopped, the gel melting time of the step 6 can be accelerated, and the recovery rate of the DNA is improved.
4. Weigh the weight of the rubber block and calculate the volume of the rubber block. When calculating the mass of the gel, the calculation was performed at 1 mg = 1 μL.
5. Add the rubber block melt DR-I Buffer to the rubber block. The dosage of DR-I Buffer is as follows:
Gel concentration DR-I Buffer usage
1% 3 gel volume
1%-1.5% 4 gel volume
1.5%-2% 5 gel volume
6. After mixing uniformly, heat at 75 ° C and melt the rubber block (low melting point agarose gel only needs to be heated at 45 ° C). At this point, the mixture should be intermittently shaken to allow the rubber block to melt sufficiently (about 6 to 10 minutes).
Note: The rubber block must be fully melted, otherwise it will seriously affect the DNA recovery rate.
7. Add 1/2 volume of DR-II Buffer in the amount of DR-I Buffer to the above-mentioned gel melt, and mix them evenly. When a DNA fragment of less than 400 bp is isolated, a final concentration of 20% isopropanol should be added to the solution.
8. Place the Spin Column in the kit on the Collection Tube.
9. Transfer the solution from Run 7 above to the Spin Column, centrifuge at 12000 rpm for 1 min, and discard the filtrate.
Note: If the filtrate is added to the Spin Column and centrifuged once, the DNA recovery rate can be improved.
10. Add 500 μL of Rinse A to the Spin Column, centrifuge at 12000 rpm for 30 s, and discard the filtrate.
11. Add 700 μL of Rinse B to the Spin Column, centrifuge at 12000 rpm for 30 s, and discard the filtrate.
12. Repeat step 11.
13. Place the Spin Column on a new 1.5 mL centrifuge tube and add 25 μL of sterile distilled water or Elution Buffer to the center of the Spin Column membrane and let stand for 1 min at room temperature.
Note: The use of sterilized distilled water or Elution Buffer heated to 60 ° C is beneficial to improve the elution efficiency.
14. The DNA was eluted by centrifugation at 12,000 rpm for 1 min.
The purified DNA fragment of interest was stored at a low temperature of -20 °C.
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