Explain the principle of identification and purification of PCR products

Principle of experiment

Agarose gel electrophoresis identifies PCR amplification products. (The principle refers to agarose gel electrophoresis to detect DNA experiments)

In this experiment, TaKaRa's Agarose Gel DNA Purification Kit was used, which is a kit for recovering and purifying DNA fragments from an agarose gel. The kit uses a unique gel thawing system, combined with DNA preparation membrane technology, featuring high efficiency, speed, and convenience. The full set of operations can be completed in just 30 minutes. Up to 10 μg of DNA fragment (100 bp to 30 kb) can be purified using this kit at a time, and the recovery rate is as high as 50 to 80%. The DNA fragment recovered and purified by the kit is of high purity and good integrity, and can be directly used for various molecular biological experiments such as ligation reaction, PCR amplification and DNA sequencing.

The DNA fragment recovered by the gel purification kit was stored at -20°C at low temperature, which delayed the degradation of DNA.

Experimental reagent

PCR product

2. Agarose Gel DNA Purification Kit (TaKaRa)

3. Agarose

4. 1×TAE

5. 6×Loading Buffer

6. DNA Marker 2000

Laboratory equipment

Agarose gel electrophoresis system

2. UV Transmissometer

Desktop centrifuge

4. Pipette gun (with 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. Make an agarose gel using 1×TAE buffer and then run the target DNA on agarose gel. (See agarose gel electrophoresis for DNA test)

2. Cut the agarose gel containing the target DNA (approximately 600 bp) under a UV lamp and blot the liquid on the gel surface with a paper towel. At this point, care should be taken to remove the gel that does not contain the DNA of interest, minimize the gel volume, and increase the DNA recovery.

Note: Be careful not to expose DNA to UV light for a long time to prevent DNA damage.

3. Cut the rubber block. After shredding the plastic block, the melting time of the plastic block in operation step 6 can be accelerated, and the recovery rate of DNA can be increased.

4. Weigh the mass of the glue and calculate the volume of the glue. When calculating the volume of the plastic block, calculate it with 1 mg=1 μL.

5. Add DR-I Buffer to the glue block. The amount of DR-I Buffer is shown in the following table:

Gel Concentration DR-I Buffer Usage

1% 3 gel volume

1%-1.5% 4 gel volumes

1.5%-2% 5 Gel Volumes

6. Heat evenly after mixing at 75°C to melt the block (low melting point agarose gel heats only at 45°C). At this point, the mixture should be intermittently oscillated to fully melt the gel (about 6 to 10 minutes).

Note: The plastic block must be fully melted, otherwise it will seriously affect the DNA recovery rate.

7. Add 1/2 volume of DR-II Buffer of DR-I Buffer to the above melted melt and mix well. When a DNA fragment smaller than 400 bp is isolated, an additional 20% of isopropanol should be added to the solution.

8. Place the Spin Column in the kit on the Collection Tube.

9. Transfer the solution of procedure 7 above to the Spin Column, centrifuge for 1 min at 12000 rpm, and discard the filtrate.

Note: If the filtrate is added to the Spin Column and centrifuged once, the DNA recovery can be increased.

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 again.

13. Place the Spin Column in a new 1.5 mL centrifuge tube. 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 heated distilled water or Elution Buffer heated to 60°C will improve the elution efficiency.

14. Elute DNA by centrifugation at 12000 rpm for 1 min.

The purified DNA fragment was stored at -20°C in a low temperature.

Other

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