Magnetic particle-based purification of PCR products
Magnetic particle-based purification of PCR products
MagneSil is a paramagnetic silica particle that acts as a mobile solid-phase material for capturing double-stranded DNA fragments. The target complexes adhering to the silica particles are washed to remove residual contamination and purify the target DNA.This experiment is from PCR Laboratory Guide (2nd edition) by Kang Seed and Lijia Qu.
Operation method
Magnetic particle-based purification of PCR products
Materials and Instruments
Ethanol PCR Sample Move I. Materials For more product details, please visit Aladdin Scientific website.
Automated Positioners Pipette Tips Liquid Handling Automated Mechanisms Separation Units Plate Seals PCR Purification Systems
1. Buffers, solutions and reagents
Ethanol, 80%
2. Nucleic acids and oligonucleotides
PCR samples in 96-well plates (no more than 100ul/well)
3. Specialized equipment
3 single-unit laboratory autopositioners (BeckmanCoulter)
4 sets of P250 cartridge pipette tips (BeckmanCoulter)
4X4 position laboratory autopositioner (BeckmanCoulter)
Automatic Positioner for 96-well pipette tips for washing experiments (BeckmanCoulter)
Automatic Positioner for Heating and Cooling Experiments (BeckmanCoulter)
Automated machinery for liquid handling, e.g. BiomekFx or Biomek2000 Automated Laboratory Workstations (BeckmanCoulter)
MagnaBot96 magnetic separation device (Promega)
Non-permeable flat seals
Cyclotron Oscillation Automatic Laboratory Positioner (BeckmanCoulter)
Automatic Laboratory Positioner for Tip Loading and Unloading (BeckmanCoulter)
Plate Clamp 96 (Promega)
4. Other
WizardMagneSilPCR Purification System [Promega; includes MagneSilYellow (paramagnetic particles), wash solution, nuclease-free water, and collection plate]
II. Methods
The steps and timings in the following protocol are provided for the use of the MagneSilPCR Purification System in the development of automated robotic processing, and the steps, as well as the volumes of reagents used, can also be used in manual processing. The system is suitable for the purification of PCR products from 100ul samples.
1. DNA Binding
(1) Starting with the PCR samples stored in a 96-well plate, adjust the volume of each sample to 100ul with nuclease-free water.
(2) Vibrate the MagneSil pellet to resuspend it sufficiently, and then add 100ul of it to each well.
(3) Pump up and down 4 times to mix the contents of the wells.
(4) Incubate at room temperature for 2 min, incubate for 1 min, and then pump up and down 4 times to mix.
(5) Transfer the mixed sample to a 96-well collection plate and place the plate on a MagnaBot96 magnetic separation device to magnetize the particles.
(6) Discard the supernatant.
2. Wash
(1) Remove the collection plate containing paramagnetic particles from the MagnaBot96 Magnetic Separation Unit and add 200ul of Wash Solution to each well.
(2) Pump up and down 4 times to mix the contents of the wells.
(3) Place the collection plate on the MagnaBot96 Magnetic Separation Unit to magnetize the particles.
(4) Discard the supernatant.
(5) Remove the collection plate from the MagnaBot96 Magnetic Separation Device and add 200ul of 80% ethanol to the wells.
(6) Pump up and down 4 times to mix the contents of the wells.
(7) Place the collection plate on the MagnaBm96 Magnetic Separation Device to magnetize the particles.
(8) Discard the supernatant.
(9) Repeat steps (5)-(8) with 200ul of 80% ethanol, making 2 ethanol washes.
(10) Leave the plate to dry on the MagnaBot96 magnetic separator for 5?10 min, and aspirate all residual ethanol from the wells.
3. DNA elution
(1) Remove the plate from the MagnaBot96 Magnetic Separator, add lOOul of nuclease-free water to elute the PCR product from the paramagnetic pellet, pump up and down four times to mix the wells, and incubate for 2 min at room temperature.
(2) Place the collection plate on a MagnaBot96 magnetic separator to magnetize the pellet.
(3) Transfer the eluted PCR product to a clean collection plate.
(4) If the eluate still carries particles, place the collection plate on the MagnaBot96 Magnetic Separation Unit and transfer the eluate to a clean collection plate, or alternatively place the collection plate on the MagnaBot96 Magnetic Separation Unit while transferring the sample from the collection plate.
(5) Seal the plate tightly with a non-permeable plate seal and store at 4°C for 1?2 days or -2°C for long-term storage.