Silica membrane-based purification of PCR products
Silica membrane-based purification of PCR products
The Wizard SV96 PCR Purification System provides a membrane-based method for high-yield PCR purification from 96-well plates. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Silica membrane-based purification of PCR products
Materials and Instruments
Ethanol PCR Sample Move I. Materials For more product details, please visit Aladdin Scientific website.
Vacuum unit Vacuum pump Vacuum waste collection unit Vacuum tube PCR purification system
1. Buffers, solutions and reagents
Ethanol, 80% (75 ml/plate; prepared before use)
2. Nucleic acids and oligonucleotides
PCR samples in 96-well plate (20?lOOul per sample)
3. special equipment
Vac-man 96 vacuum unit (Promega) or similar vacuum unit
Vacuum pump capable of generating 15?20in (lin=2.54 cm) Hg pressure
Vacuum waste collection unit (1L size)
Vacuum hose
4. Other
WizardSV96PCR Purification System (Promega; includes binding plate, collection plate, DNA elution plate, membrane binding solution, and nuclease-free water)
II.
1. Prepare the vacuum unit as shown in Figure 9-8. Place the binding plate on the bottom of the vacuum unit. To ensure that the wells on the sample plate correspond to the wells on the binding plate, place a vacuum marking line on the side of the binding plate with the numbered markings toward the end of the vacuum port in the vacuum device, and attach a vacuum marking line to the vacuum port at the base of the vacuum device. 
2. Add the same volume of Membrane Binding Solution to each PCR Sample in the 96-well plate (e.g., 100ul of Membrane Binding Solution per lOOul of PCR Sample).
3. Aspirate the mixture and transfer all samples to the wells of the binding plate placed on top of the vacuum unit for 1 minute at room temperature.
4. Evacuate until the sample passes through the binding plate (~30s), release the vacuum.
5. Add 200ul of 80% ethyl libation to each well of the Binding Plate and incubate for 1 min at room temperature, evacuate until the ethyl libation passes through the Binding Plate (approx. 30s), release the vacuum.
6. Repeat step 5 for 3 washes with 200ul of 80% ethanol.
7. In the last wash, when the wells of the binding plate become empty, continue vacuuming for 4 min to dry the binding matrix.
8. Turn off the vacuum, loosen the vacuum line at the bottom of the vacuum unit and allow it to be sucked violently into its empty port located in the gasket of the vacuum unit. Remove the binding plate from the base of the vacuum unit and pat it on a clean paper towel to dry and remove any residual ethanol.
9. Place the 96-hole, U-bottomed collection plate in the bottom bed of the Vacuum Unit with the Vacuum Unit Gasket installed on top, with the numbered side of the U-bottomed plate facing the vacuum port.
10. Place the bond plate on top of the vacuum unit gasket and the collection plate as shown in Figure 9-8. The tip of the bond plate must be aligned with the center of the holes in the collection plate and both plates must be oriented the same way. Add l00ul of nuclease-free water to each well of the binding plate, incubate for 1 min at room temperature, and evacuate until the solution passes through the plate (approximately 1 min).
11. Release the vacuum and remove the Binding Plate, carefully removing the vacuum unit gasket to ensure that the Collection Plate remains in the vacuum unit bed. If small droplets remain on the walls of the collection plate, centrifuge the plate for a short time to concentrate the small droplets to the bottom of the wells. The volume of the eluate may vary and is approximately 75 ml.