A Magnetic Particle-Based M...

A Magnetic Particle-Based Method for Purifying PCR Products from Solu... http://cshprotocols.cshlp.org/cgi/content/full/2006/1/pdb.prot4100?print=true
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Cite as: Cold Spring Harb. Protoc.; 2006; doi:10.1101/pdb.prot4100
Protocol
A Magnetic Particle-Based Method for Purifying PCR Products from Solution
Craig Smith, Paul Otto, Rex Bitner, and Gary Shiels
This protocol was adapted from "DNA Purification," contributed by Craig Smith, Paul Otto, Rex Bitner,
and Gary Shiels in Chapter 9, PCR Primer, 2nd edition (eds. Dieffenbach and Dveksler). Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2003.
INTRODUCTION
The MagneSil system can selectively isolate PCR products that are more than 150-bp long from primers and primer
-dimers. The technology can be used with a number of robotic workstations, including Beckman Coulter’s Biomek
2000 and FX Laboratory Automation Workstations. The procedure can also be carried out manually. Typical recovery
is more than 80% for a 1-kb product with negligible carryover of primers or nucleotides.
MATERIALS
Reagents
Ethanol, 80%
PCR samples in 96-well format (up to 100 µl/well)
Wizard MagneSil PCR Clean-Up System (Promega; includes MagneSil YELLOW [paramagnetic particles], wash
solution, nuclease-free H 2 O, and collection plates)
Equipment (for automated procedure)
Liquid-handling robot such as the Biomek FX or Biomek 2000 Laboratory Automation workstation (Beckman Coulter),
including :
Three single-position labware ALPs (automated labware positioners) (Beckman Coulter)
Four P250 tip rack assemblies (Beckman Coulter)
4X 4-position labware ALPs (Beckman Coulter)
96-well Tip Wash ALP (Beckman Coulter)
Heating and Cooling ALP (Beckman Coulter)
Orbital Shaker ALP (Beckman Coulter)
Tip Loader ALP (Beckman Coulter)
MagnaBot 96 Magnetic Separation Device (Promega)
Nonpermeable plate sealer
Plate Clamp 96 (Promega)

A Magnetic Particle-Based Method for Purifying PCR Products from Solu... http://cshprotocols.cshlp.org/cgi/content/full/2006/1/pdb.prot4100?print=true
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METHOD
The following protocol contains the steps and timing that are intended for use of the MagneSil PCR Clean-Up System
in the development of an automated process using a robot. These same steps, including the specified reagent
volumes, may also be used to perform the procedure manually. This system was designed to purify PCR products
from 100-µl sample volumes.
DNA Binding
1. Begin with completed PCR samples in a 96-well plate. Adjust the volume of each sample to 100 µl with
nuclease-free H 2 O.
2. Resuspend the MagneSil paramagnetic particles thoroughly by shaking and add 100 µl to each well.
3. Mix the contents of each well by pipetting up and down four times.
4. Incubate for 1 minute at room temperature and mix again. Incubate for a further minute at room
temperature.
5. Transfer the mixed samples to a 96-well collection plate. Place the plate onto the MagnaBot 96 magnetic
separation device to separate the phases.
6. Remove and discard the supernatant.
Washing
7. Remove the collection plate containing the paramagnetic particles from the separation device and add 200
µl of wash solution to each well.
8. Mix the contents of each well and return the plate to the separation device. Remove and discard the
supernatant.
9. Remove the plate from the separator and add 200 µl of 80% ethanol to each well.
10. Mix the contents of each and return the plate to the separation device. Remove and discard the
supernatant.
11. Repeat Steps 7-10 with 200 µl of 80% ethanol, for a total of two ethanol washes. Then leave the plate to
dry on the separator for 5-10 minutes. Remove any residual ethanol from the wells by pipetting.
Elution of DNA
12. Remove the plate from the separator and add 100 µl of nuclease-free H 2 O to elute the PCR products from
the paramagnetic particles. Mix each well by pipetting up and down four times. Incubate for 2 minutes at room
temperature.
13. Place the collection plate onto the separation device and transfer the eluted PCR products to a clean
collection plate.
14. If particles have been carried over with the eluate, place the new collection plate on the separator and
transfer the eluate to another clean collection plate. Alternatively, keep the collection plate on the separator
when withdrawing samples.
15. Seal the plate tightly with nonpermeable plate sealer and store at 4°C for 1-2 days or at -20°C for
long-term storage.
Copyright © 2006 by Cold Spring Harbor Laboratory Press. Online ISSN: 1559-6095 Terms of Service All rights reserved. Anyone
using the procedures outlined in these protocols does so at their own risk. Cold Spring Harbor Laboratory makes no representations
or warranties with respect to the material set forth in these protocols and has no liability in connection with their use. All materials
used in these protocols, but not limited to those highlighted with the Warning icon, may be considered hazardous and should be used
with caution. For a full listing of cautions, click here.

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