We need to wash the PCR eight-tube kit after use. There are a few ways to clean the PCR Octal Kit. What should I pay attention to when using it?
Principle of the experimental method: Silica gel membrane binds to DNA under high-salt conditions, and separates from DNA under low-salt conditions. Primers, mononucleotides, enzymes, mineral oil, salt ions, etc. are separated out in the DNA-containing wash solution as they do not have similar properties to DNA.
Experimental materials: PCR products, reagents, kits, PCR cleaning kits, instruments, consumables, 96-well DNA preparation plates, 96-well deep-well plates, 96-well V-shaped plates
1. The PCR eight-tube manufacturer talks about the composition, storage and stability of the kit
1. Instructions, consumables: 96-well DNA preparation plate, 96-well 1.6ml deep-well plate, 96-well V-shaped plate.
2. Buffer PCR-A: DNA binding solution. Store tightly closed at room temperature. If precipitation occurs, it should be dissolved in a warm bath at 65°C and cooled to room temperature before use.
3. Buffer W2 concentrate: remove the salt solution. Before use, add ethanol to the volume specified on the bottle, mix well, and store at room temperature. 100% ethanol or 95% absolute ethanol can be used.
4. Eluent: 2.5 mM Tris-HCl, pH 8.5, stored in a sealed state at room temperature.
2. The manufacturer of PCR eight-coupled tubes talks about the operation steps
Users can choose negative pressure or centrifugation.
A. Negative pressure method
1. Correctly connect the negative pressure device, place the 96-well DNA preparation plate on the negative pressure device; add 3 times buffer PCR-A to the PCR, enzyme digestion, enzyme labeling or sequencing reaction solution (if the buffer PCR-A is less than 100 μl, add 100 μl); mix well and 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.
2. Add 0.3 ml of buffer W2 and absorb the solution. Wash twice with 0.3 ml of buffer W2 in the same manner.
Confirm addition of absolute ethanol to the indicated volume of Buffer W2 concentrate on the reagent bottle.
3. Maintain the negative pressure and extract the 96-well DNA preparation plate for 10 minutes.
4. Grind the 96-well DNA preparation plate 6 times on the long fibrous tissue with the drainage tube facing down.
5. Place the 96-well DNA preparation plate on a 96-well V-shaped plate, add 25-30ul of water or elute to the center of the membrane, and let stand for 1 minute at room temperature. DNA was eluted by centrifugation at 3 000 x g for 5 min.
B. Centrifugal
1. Add 3 volumes of Buffer PCR-A (if Buffer PCR-A is less than 100 μl, add 100 μl) to PCR, digestion, enzyme labeling or sequencing reactions; mix and transfer to 96-well DNA in 96-well preparation plate. Place the DNA preparation plate in a 96-well 1.6 ml deep well plate, centrifuge at 1000 x g for 1 min, and discard the filtrate.
2. In a 96-well DNA preparation plate, add 0.3 ml of buffer W2, centrifuge at 1000×g for 1 minute, and discard the filtrate. Wash again in the same manner with 0.3 ml of buffer W2. Confirm addition of absolute ethanol to the indicated volume of Buffer W2 concentrate on the reagent bottle.
3. Place the 96-well DNA preparation plate in a 96-well 1.6ml deep-well plate and centrifuge at 3 000×g for 10 minutes.
4. Place the 96-well DNA preparation plate in a clean 96-well V-shaped bottom plate, add 25-30ul of water or elute to the center of the membrane, and let stand for 1 minute at room temperature. DNA was eluted by centrifugation at 3 000 x g for 5 min.
PCR octal tube manufacturers talk about matters needing attention:
1. Heating the eluent or water to 65°C can help improve the elution efficiency.
2. DNA molecules are acidic, it is recommended to store in 2.5 mM Tris-HCl, pH 8.5 eluate.
