Oct 24, 2022
Automated 96-well PCR Purification
- Ariel Rabines1,
- Rob Lampe2,
- Andrew E Allen2
- 1J. Craig Venter Institute;
- 2Scripps Institution of Oceanography
Protocol Citation: Ariel Rabines, Rob Lampe, Andrew E Allen 2022. Automated 96-well PCR Purification. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpr19jvzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 31, 2020
Last Modified: October 24, 2022
Protocol Integer ID: 37637
Keywords: PCR purification, Eppendorf epMotion, bead cleanup, automatic, well pcr plate format with ampure xp bead, well pcr purification, pcr cleanup, automated pcr cleanup, well pcr purification this protocol, ampure xp bead, pcr product, pcr plate, well pcr plate format, pcr, bead ratio, bead magnet, thermomixer module, purification, bead
Abstract
This protocol describes a fully-automated PCR cleanup in a 96-well PCR plate format with AMPure XP beads on an Eppendorf epMotion. Multichannel pipetting with the 300 uL and 50 uL pipettes is used. It is designed to use the thermomixer module; however, this can be replaced by modifying the program to mix by pipetting. The bead magnet used in this program is a Alpaqua Low Elution Magnet Plate. We also use Eppendorf twin.tec PCR plates (LoBind, skirted, 150uL).
IMPORTANT: The program is currently set to add 22 µL of water to each well followed by 65 µL beads. This results in an approximately 1:1.5 bead ratio with a starting volume of 22 µL PCR product. Modify the first step accordingly with your starting volume and desired bead ratio.
The manufacturer's instructions are found here: https://www.beckmancoulter.com/wsrportal/techdocs?docname=B37419
The epMotion program is attached here.
Attachments
Materials
Agencourt AMPure XP beads
Eppendorf twin.tec® PCR 96-well plate, skirtedEppendorfCatalog #951020401
Molecular grade water nuclease-free
100% Molecular grade ethanol
Equipment
LE Magnet Plate
NAME
Alpaqua
BRAND
A000350
SKU
Troubleshooting
Take aliquot of beads out of refrigerator and allow to warm to room temperature.
Start program and input the numer of samples you have. It will then tell you the minimum volumes for each reagent. The positions in the reservoir rack are:
1. Molecular grade H2O
3. Beads
5. 70% EtOH
7. Elution buffer (e.g. H2O, TRIS-Acetate (10 mM pH 8.0), or TE Buffer (10 mM Tris-Acetate pH 8.0, 1 mM EDTA))
You can use the level sensor to detect volumes or measure them with a serological pipette. Do not start the program until all of your items are in the correct positions.
Mix beads well until they appear homogenous.
Prepare fresh 70% EtOH.
From the Agencourt XP beads manual: "Fresh 70% ethanol should be prepared for optimal results. There is also miscibility involved with ethanol and water. For example, measuring out 70 mL of ethanol and topping off to 100 mL with water will generate ~65% ethanol. Measuring 70 mL ethanol and 30 mL water separately, then combining them will generate ~95 mL of 70% ethanol."
Place reagents, your PCR plate, and a new clean plate on the epMotion.
Materials layout:
Run the program. It will take approximately 1 hour for a full plate.