eLabNotebook - Promega's Automated Wizard* MagneSil* 384-Well PCR* Clean-Up System on the Biomek® FX

eLabNotebook > Nucleic Acid Prep & Purification > PCR Cleanup > Promega MagenSil* 384-well
Clean-up Biomek® FX

Promega's Automated Wizard* MagneSil* 384-Well PCR* Clean-Up System on the Biomek® FX

Promega Corporation – www.promega.com

Before You Begin

Materials to Be Supplied by the User:

• ethanol, 80%, 75ml per 384-well plate

• nuclease-free water, 65ml per 384-well plate

• 2 Microseal* 384-well, polypropylene, microplates V.2.0 (MJ Research Inc., Part# MSP-3842) (Note: This method was written and tested using an M.J. Research, Inc. 384-well PCR plate for sample and final elution plates.Other 384-well PCR plates may be substituted, with the understanding that modifications to the method will be required.)

• 2 Greiner 384-well, polystryrene microplates (Promega Cat.# V5291)

• MagnaBot* 384 Magnetic Separation Device (Promega Cat.# V8241)

• Turbulator2* Reservoir (ACME Autmation Cat.# G5058)
(Note: This includes polypropylene reservoir, silicone tubing, 1/2”, Gast vacuum pump, DOA-P104-AA (www.acme-automation.com)

A. Preparation of Solutions

Turbulator2* Reservoir Notes: The Turbulator2* reservoir must be hooked up to a vacuum pump before the MagneSil* YELLOW is added. This is done by attaching the tubing from the Turbulator2* to a vacuum pump, turning the vacuum pump “on,” and adjusting the pressure between 2 and 5 inches Hg. When the Turbulator2* reservoir is placed onto the deck, press the reservoir firmly into place. Ensure that the tubing from the Turbulator2* is not over the light curtain on the Biomek® FX workstation.

MagneSil* YELLOW PMPs Notes: Add 100ml of MagneSil* YELLOW PMPs to the Turbulator2* reservoir before running the method. Not all of the resin will be used during the method. This volume is necessary to eliminate splattering of the resin while the Turbulator2* reservoir is running. Unused MagneSil* YELLOW PMPs may be poured back into the MagneSil* YELLOW bottle after the method is complete. This is done by pouring the resin into a Beckman upsidedown tip lid while the Turbulator2* reservoir is still running. The resin can then be poured back into the bottle.

Resuspension and dispensing the PMPs are explained in the comment lines of 384-well PCR clean-up electronic method for the Biomek® FX Workstation.

B. Sample Preparation Before Automated Processing

Ensure that the 20µl volume of unpurified PCR is at the bottom of the well of the 384-well sample PCR plate by centrifuging the sample plate briefly at high speed.

C. Initial Deck Configuration for the Beckman Biomek® FX Workstation

(Click to Enlarge)
Figure 1. Biomek® FX initial deck configuration.

Beckman Biomek® FX Deck Layout. This is an example of a MagneSil* PCR Clean-Up System deck layout on a Biomek® FX.Your specific deck layout may be different depending on your Biomek® FX configuration.

ALP Name	Part Siting on ALP
Tip Loader	AP_384 30µl Biomek* FX tips
P1	AP_384 30µl Biomek® FX tips
P2	Turbulator2* containing 100ml of MagneSil* YELLOW PMPs
P3	384-well PCR plate containing 20µl of PCR amplifications/well
P4	Beckman Coulter upside-down tip box lid containing 65ml of Wash Solution
P5	Swap Space. This is not a specific piece of labware. It is a place holder used to swap tips during the method.
P6	W1 Labware Reserved Spot/Empty 384-well Greiner plate (polypropylene)
P7	Beckman Coulter upside-down tip box lid containing 75ml of 80% ethanol
P9	MagnaBot* 384 Magnetic Separation Device
P10	Beckman Coulter upside-down tip box lid containing 65ml of nuclease-free water
P12	Empty 384-well Greiner plate (polystyrene)
P13	Empty 384-well PCR plate (polypropylene)
P8, P11, P14–18 Empty.	Positions only necessary for multiple plate methods.
TR1	Left Trash ALP. Only necessary for multiple plate methods.
Tip Wash ALP	384-well Tip Wash Station

D. Pre-Run Beckman Biomek® FX Specific Recommendations

The Beckman Biomek® FX automated platform allows users the flexibility to configure the robot’s deck according to need. Because of this flexibility in deck configuration, an end-user’s deck may differ from the deck used for writing the Biomek® FX method. Therefore, mapping an imported method onto the enduser’s deck configuration is generally necessary. Follow the instructions provided: Biomek® FX Deck Mapping
(www.promega.com/automethods/beckman/biomekfx)

Prior to the first run of the MagneSil* 384-Well PCR Clean-Up method on the Beckman Biomek® FX workstation, you should ensure that the deck has been properly framed. Failure to do so may result in bent tips during the run. Position the AP 384 30µl tip box on the Tip Loader so that it is pushed against the front left corner of the ALP. This will ensure proper seating of the tips into the mandrels of the 384 Multichannel Pod.

E. Description of Automated Wizard® MagneSil™ 384-Well PCR Clean-Up

This overview describes general liquid handling steps required for 384-well clean-up of a 20µl PCR sample and can be adapted to a variety of automated liquid handling robots. For additional information about adapting this protocol to liquid handling robots other than those referenced above, please See section VII.

A. Binding DNA to MagneSil* YELLOW PMPs

	1.	*MagneSil YELLOW PMPs Transfer.** A volume of MagneSil* YELLOW PMPs (equal to the PCR sample volume) is transferred from the Turbulator2* reservoir to the processing plate. For example, if purifying a 20µl PCR, 20µl of MagneSil* YELLOW PMPs is transferred to the processing plate.
	2.	PCR Transfer. Twenty microliters is transferred from the PCR sample plate to the processing plate containing the MagneSil* YELLOW PMPs and mixed by pipetting.Ten microliters of the mixed PMPs and amplification mix is transferred back to the sample PCR plate and mixed in the PCR plate by pipetting. The PMPs and amplification mix are transferred back to the processing plate. This step ensures that all of the PCR sample is mixed with the PMPs for optimal recovery of unpurified PCR products.
	3.	Binding Mixes. The PMPs and PCR sample are mixed by pipetting (25µl volume) to bind the amplification products to the PMPs. The robot pauses for 2 minutes. A second mix is performed to increase binding efficiency.
	4.	On-Magnet Mix and Supernatant Removal. The processing plate is transferred to the MagnaBot* 384 Magnetic Separation Device. The robot pauses for 20 seconds to capture PMPs. The liquid is mixed by pipetting to ensure complete capture of PMPs to the sides of each well. The robot pauses for 5 seconds. The entire supernatant is transferred from the processing plate that is sitting on the MagnaBot* 384 device to the 384-well Tip Wash Station. Removing all of the supernatant at this step is critical.

B. Washing MagneSil* YELLOW PMPs

	1.	Processing Plate Transfer. The processing plate is transferred from the MagnaBot* 384 device to the W1 labware reserved spot.
	2.	Wash Solution Transfer. Forty microliters (2X the PCR sample volume) of Wash Solution is transferred to the processing plate, using two transfers of 20µl each from the Wash 1 reservoir to the processing plate. When using the Biomek® FX workstation, a tip touch on the liquid in the 384-well Tip Wash Station is performed to remove droplets.
	3.	Wash Mixes. PMPs and PCR samples are mixed by pipetting (25µl volume) to wash. The robot pauses for 2 minutes and then repeats the mix to increase the washing efficiency.
	4.	On-Magnet Mix and Supernatant Removal. The processing plate is transferred to the MagnaBot* 384 device. The robot pauses for 20 seconds to capture PMPs to the sides of each well. The liquid is mixed by pipetting to ensure complete capture of PMPs. The robot pauses for 5 seconds. All of the supernatant is transferred from the processing plate sitting on the MagnaBot* 384 device to the 384-well Tip Wash Station.

C. Ethanol Wash of MagneSil* YELLOW PMPs

	1.	Processing Plate Transfer.The processing plate is transferred from the MagnaBot* 384 device to the W1 labware reserved spot.
	2.	80% Ethanol Transfer.Twenty microliters of 80% ethanol (1X the PCR sample volume) is transferred to the processing plate. The Biomek® FX workstation performs a tip touch on the liquid in the 384-well Tip Wash Station to remove ethanol droplets.
	3.	Wash Mixes.PMPs and PCR samples (15µl volume) are mixed by pipetting to wash. The robot pauses for 2 minutes then repeats the mix with pipette tips to increase the washing efficiency.
	4.	On-Magnet Mix and Supernatant Removal.The processing plate is transferred to the MagnaBot* 384 device. There is a 20-second pause to capture PMPs to the sides of each well. PMPs and PCR samples are mixed by pipetting to ensure complete capture of PMPs. The robot pauses for 5 seconds. All of the supernatant from the processing plate sitting on the MagnaBot* 384-device is transferred to the 384-well Tip Wash Station. 5. Wash Step Repeat. Repeat steps 1–4 for a total of 3 washes of the PMPs with 80% ethanol.

D. Drying the MagneSil* YELLOW PMPs

	1.	Final Supernatant Removal. The robot pauses for 30 seconds to allow time for any residual ethanol to move to the bottom of each well. Twenty-five microliters (1.25X the PCR sample volume) is transferred from the processing plate to the 384-well Tip Wash Station. This step is necessary to ensure that all possible ethanol has been removed from each well of the processing plate.
	2.	*Dry MagneSil YELLOW PMPs.** The robot pauses with the processing plate on the MagnaBot® 384 device for a total of 420 seconds. This allows any ethanol remaining on the PMPs, or in the plate, to evaporate.
	3.	Wash Sample and Reagent Tips. While the processing plate is paused, the robot rinses the sample and reagent tips in the 384-well Tip Wash Station to remove any ethanol or PMPs from the tips before the elution step.

E. Eluting DNA from MagneSil™ YELLOW PMPs

	1.	Processing Plate Transfer. The processing plate is transferred from the MagnaBot® 384 device to the W1 labware reserved spot.
	2.	Elution Water Transfer. Fifty microliters (2X the PCR sample volume) of elution water is transferred to the processing plate.
	3.	Elution Mixes. The liquid is mixed by pipetting (25µl volume) to elute the purified amplification products from the PMPs. The robot pauses for 1 minute. A second mix is performed to increase the elution efficiency.
	4.	On-Magnet Mix and Eluate Transfer. The processing plate is transferred to the MagnaBot® 384 device. There is a 20-second pause to capture PMPs to the sides of each well. The supernatants are mixed by pipetting to ensure complete capture of PMPs. The robot pauses for 5 seconds. Fifty microliters (2.5X the PCR sample volume) is transferred from the processing plate to the empty 384-well Greiner plate or elution plate 1. The processing plate is transferred from the MagnaBot® 384 device to the W1 labware reserved spot. Elution plate 1 is transferred to the MagnaBot® 384 device. There is a 20-second pause to capture PMPs to the sides of each well. Thirty microliters (1.5X the PCR sample volume) is transferred from elution plate 1 to the empty 384-well PCR plate, or elution plate 1b.

F. General Guidelines for Adaptation to Alternative Robotic Platforms

Following the final ethanol removal, the processing plate is dried for 7 minutes. This drying is critical to remove all possible residual ethanol. The use of a heating ALP may be incorporated for the drying step. The number of mixes may need to be increased to ensure complete resuspension of the PMPs in each well of the processing plate.

The MagneSil* YELLOW PMPs used for this purification process settle rapidly over time. This method uses the Turbulator2* mixing reservoir to keep the MagneSil* YELLOW PMPs in suspension. If the MagneSil* YELLOW is dispensed into a 384-well plate prior to running the method, we recommend thoroughly mixing the MagneSil* YELLOW PMPs on the automated platform prior to dispensing to samples.

The single-plate method described above uses a single box of tips for dispensing reagents and a second for manipulating samples. If a multiple-plate method is to be developed, the method should still use 1 box of tips for all reagent dispensings but should use a separate box of tips for the manipulations of each sample plate. This setup prevents any possible cross-contamination.

* All trademarks are the property of their respective owners. Where applicable, the PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffman-LaRoche, Ltd.