DNA purification experiment method
PCR cleaning kit purification method
Method principle
The silica membrane can bind DNA under high-salt conditions, and can separate from DNA under low-salt conditions. Impurities such as primers, single nucleotides, enzymes, mineral oil, salt ions, etc. in the DNA solution are separated because they do not have properties similar to those of DNA.
Experimental Materials:
PCR products, reagents, kits, PCR cleaning kits
Instrument consumables:
96-well DNA preparation plate, 96 well deep well plate, 96-well V-shaped bottom plate
[Kit composition, storage, stability]
Consumables: 96-well DNA preparation plate, 96-well 1.6ml deep-well plate, 96-well V-shaped bottom plate.
BufferPCR-A: DNA binding solution. Airtight storage at room temperature. If precipitation occurs, it should be dissolved in a warm bath at 65°C and cooled to room temperature before use.
BufferW2concentrate: Desalt solution. Before use, add ethanol according to the volume specified on the bottle, mix well, and store in airtight at room temperature. Available with 100% ethanol or 95% absolute ethanol
Eluent: 2.5mMTris-HCl, pH8.5. Airtight storage at room temperature.
Experimental operation
A. Negative pressure method
Connect the negative pressure device correctly, and place the 96-well DNA preparation plate on the negative pressure device;
Add 3 volumes of BufferPCR-A to the PCR, restriction enzyme digestion, enzyme labeling or sequencing reaction solution (if BufferPCR-A is less than 100ul, add to 100ul);
After mixing, transfer to a 96-well DNA preparation plate, turn on and adjust the negative pressure to -25-30 inches Hg, and slowly aspirate the solution in the plate.
Add 0.3ml BufferW2 and suck the solution. In the same way, wash twice with 0.3ml BufferW2.
Keep the negative pressure and aspirate the 96-well DNA preparation plate for 10 minutes.
Tap the 96-well DNA preparation plate on a long-fibrous paper towel with the flow tube facing down for 6 times.
Place the 96-well DNA preparation plate on the 96-well V-shaped bottom plate, add 25-30ul of water or Eluent to the center of the membrane, and let it stand at room temperature for 1 min. Centrifuge at 3000xg for 5 min to elute DNA.
* Make sure that absolute ethanol has been added to the BufferW2concentrate according to the specified volume on the reagent bottle.
B. Centrifugation
Add 3 volumes of BufferPCR-A to the PCR, restriction enzyme digestion, enzyme labeling or sequencing reaction solution (if BufferPCR-A is less than 100ul, add to 100ul);
After mixing, transfer to a 96-well DNA preparation plate, place the 96-well DNA preparation plate in a 96-well 1.6 ml deep-well plate, centrifuge at 1000 xg for 1 min, and discard the filtrate.
In a 96-well DNA preparation plate, add 0.3ml BufferW2, centrifuge at 1000xg for 1min, discard the filtrate, and wash again with 0.3ml BufferW2 in the same way.
Place the 96-well DNA preparation plate in a 96-well 1.6ml deep-well plate and centrifuge at 3000xg for 10 min.
Place the 96-well DNA preparation plate in a clean 96-well V-shaped bottom plate, add 25-30ul of water or Eluent to the center of the membrane, and let it stand at room temperature for 1 min. Centrifuge at 3000xg for 5 min to elute DNA.
* Make sure that absolute ethanol has been added to the BufferW2concentrate according to the specified volume on the reagent bottle.
DNA gel recovery kit purification method
Method principle
The agarose gel is dissolved in a mild buffer (DE-A solution). The protective agent in it can prevent linear DNA from degrading at high temperatures, and then the DE-B solution allows the DNA to selectively bind to the membrane. The purified DNA has high purity, and maintains fragment integrity and high biological activity. It can be directly used in molecular biology experiments such as ligation, in vitro transcription, PCR amplification, sequencing, and microinjection.
Instrument consumables:
DNA preparation tube, 2ml centrifuge test tube, 1.5ml centrifuge tube
[Kit composition, storage, stability]
BufferDE-A: Gel melting agent, containing DNA protective agent to prevent DNA degradation at high temperature. Airtight storage at room temperature.
BufferDE-B: Binding solution (to promote selective binding of DNA fragments larger than 70bp to the DNA preparation membrane). Airtight storage at room temperature. If precipitation occurs, incubate at 70°C to dissolve and cool to room temperature before use.
BufferW1: Washing solution, sealed and stored at room temperature.
BufferW2concentrate: Desalt solution. Before use, add ethanol according to the amount on the bottle, mix well, and store in airtight at room temperature.
100% absolute ethanol or 95% ethanol.
Eluent: 2.5mM Tris-HCl, pH 8.5, airtight storage at room temperature.
Experimental operation
Cut the agarose gel containing the target DNA under a UV light (cut into small pieces, do not expose to UV light for a long time to prevent DNA damage), use a paper towel to absorb the liquid on the surface of the gel and chop it up.
Calculate the gel weight (record the weight of the 1.5ml centrifuge tube in advance), and use this weight as a gel volume (for example, 100mg=100ul volume).
Add 3 gel volumes of BufferDE-A, mix well and heat at 75°C (low melting point agarose gel is heated at 40°C), mixing intermittently (every 2-3min) until the gel mass is completely melted (about 6- 8min).
Add 0.5 BufferDE-A volume of BufferDE-B and mix well; when the separated DNA fragment is less than 400bp, add 1 gel volume of isopropanol.
Steps 4-6 can choose negative pressure method or centrifugal method.
A. Negative pressure method
4A. Connect the negative pressure device correctly, and insert the DNA preparation tube into the socket of the negative pressure device. Absorb the mixed solution in step 3, transfer it to the preparation tube, turn on and adjust the negative pressure to -20-30 inches Hg, and slowly aspirate the solution in the tube.
5A. Add 0.5ml BufferW1, and suck up the solution.
6A. Add 0.7ml BufferW2, suck the solution. Wash again with 0.7ml BufferW2 in the same way.
B. Centrifugation
4B. Aspirate the mixed solution in 3, transfer it to a DNA preparation tube (placed in a 2ml centrifuge tube), and centrifuge at 12000xg for 1 min. Discard the filtrate.
5B. Put the preparation tube back into the centrifuge tube, add 0.5ml BufferW1, centrifuge at 12000xg for 30s, and discard the filtrate.
6B. Put the preparation tube back into the centrifuge tube, add 0.7ml BufferW2, centrifuge at 12000xg for 30s, and discard the filtrate. (Optional step) Wash again with 0.7ml BufferW2 in the same way, and centrifuge at 12,000xg for 1 min.
7. Place the preparation tube in a 2ml centrifuge tube and centrifuge at 12000xg for 1 min.
8. Place the preparation tube in a clean 1.5ml centrifuge tube, add 25-30ul of water or Eluent to the center of the DNA preparation membrane, and let it stand at room temperature for 1 min. Centrifuge at 12000xg for 1 min to elute DNA.
* Make sure that absolute ethanol has been added to the BufferW2concentrate according to the specified volume on the reagent bottle.
