eLabNotebook - Promega MagneSil* ONE Biomek® FX

eLabNotebook > Nucleic Acid Prep & Purification > PCR Cleanup > Promega Wizard* SV 96
Clean-up Biomek® FX

Promega's Automated Wizard* SV 96 PCR* Clean-Up System on the Biomek® FX

Promega Corporation – www.promega.com

Before You Begin

Materials to Be Supplied by the User:

• 80% ethanol or 95% ethanol (70ml/plate). Use 95% ethanol for purification of amplification products <500 bases.

• 96-well Greiner U-bottom plate (Promega Cat.# A9161)

A. Preparation of Solutions

Prepare a solution of 80% ethanol solution just before performing the automated clean-up procedure. An alcohol concentration lower than 80% will result in decreased efficiency of sample recovery. For purification of small amplification products (<500 bases), increase the ethanol wash concentration to 95% for improved efficiency of recovery.

B. Sample Preparation

Ensure that all sample is at the bottom of the 96-well PCR plate. If not, briefly spin the plate in a centrifuge to move all samples to the bottom of the wells.

C. Biomek® FX Deck Set-Up

Figure 1 shows an example of a deck layout for set-up of the SV 96 PCR Clean- Up System on the Biomek® FX.Your specific deck layout may be different depending on your Biomek® FX configuration.

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

ALP Name	Equipment
Tip Loader	200µl non-ART® Biomek® FX Tips
P1	A non-skirted 96-well PCR plate in a Plate Clamp sitting on a Plate Stand.
P2	200µl non-ART® Biomek® FX Tips
P3	200µl non-ART® Biomek® FX Tips
P4	Pyramid bottom reservoir plate containing Xml 80% (or 95%) ethanol (see Section III.A)
P5	Greiner 96-well U-bottom plate containing Xµl Membrane Binding Solution per well
P6	Swap spot
P7	Pyramid bottom reservoir plate containing Xml nuclease-free water.
P8	96-well Elution Plate
SPE ALP	Vacuum filtration manifold base, elution spacer, 36mm collar, SV 96 Binding Plate

D. Biomek® FX Specific Pre-Run Recommendations

The Biomek® FX automated platform allows users the flexibility to configure the robot's deck according to need. Because of this flexibility, it is likely that the deck used for writing a Biomek® FX method will differ from an end-user's deck. Therefore, it is generally necessary to map an imported method onto an enduser's deck configuration. To map an imported method onto your deck, please follow the instructions provided in the document Biomek® FX Deck Mapping (www.promega.com/automethods/beckman/biomekfx/default.asp).

Prior to the first run of the SV 96 PCR Clean-Up method on the Beckman Biomek® FX, check all Gripper moves to ensure that the vacuum manifold disassembly and reassembly for elution is correct. We have found that Gripper moves for vacuum manifold disassembly and reassembly differ for each Biomek® FX instrument. Failure to do this Gripper test evaluation may result in vacuum manifold disassembly and reassembly failing and may even result in a Gripper crash. To check Gripper moves, please follow the instructions provided in the document: Evaluation of Biomek® FX SV 96 Method Gripper Moves (www.promega.com/automethods/beckman/biomekfx/default.asp).

Evaluation of Biomek® FX SV 96 Purification Method Gripper Moves requires the Beckman Biomek® FX Grip Test method. Please inquire for this method.

E. Description of Automated SV 96 PCR Clean-Up

This overview describes the general liquid handling steps required for automated SV 96 PCR Clean-Up. The procedure can be adapted for performance on a variety of automated liquid handling robots. Additional information for adaptation to liquid handling robots other than the Biomek® 2000 and Biomek® FX is provided in Section VII.

	1.	Preparation of samples for binding to SV 96 Binding Plate. An equal volume of Membrane Binding Solution is transferred to each sample in a 96-well PCR plate and mixed using pipet tips.
	2.	Transfer samples to SV 96 Binding Plate. The sample (with Membrane Binding Solution added) is transferred to the SV 96 Binding Plate that is sitting on top of the vacuum manifold apparatus.
	3.	Bind nucleic acids to SV 96 Binding Plate. Once all the sample has been transferred to the SV 96 Binding Plate, the vacuum is applied and sample is drawn through the Binding Plate for 30 seconds. During this step, nucleic acids bind to the SV 96 Binding Plate.
	4.	Wash #1. Two hundred microliters of 80% (or 95%, see Section III.A) ethanol is dispensed to each well of the SV 96 Binding Plate. After a one-minute pause, the vacuum is applied for 30 seconds. The wash solution is pulled through the SV 96 Binding Plate.
	5.	Washes #2 & 3. Step 4 is repeated for a total of three washes of the SV 96 Binding Plate.
	6.	Drying/removal of residual alcohol. The vacuum remains on for four more minutes to remove any residual ethanol from the SV 96 Binding Plate.
	7.	Preparation for elution. After the final vacuum step there is a one-minute pause to allow for complete vacuum ventilation before disassembly and reassembly for the final elution step. A gripper tool disassembles the vacuum manifold stack by removing the SV 96 Binding Plate and vacuum collar from the vacuum manifold to holding position. The gripper then moves a 96-well Elution Plate into the vacuum manifold. The gripper then reassembles the vacuum manifold stack by moving the SV 96 Binding Plate and vacuum collar back onto the vacuum manifold over the top of the Elution Plate (Figure 2).
	8.	Elution of purified amplification products. One hundred microliters of Nuclease-Free Water is transferred from the reservoir to each well of the SV 96 Binding Plate. After a one-minute pause, the vacuum is applied. Nuclease-Free Water is pulled through the SV 96 Binding Plate, eluting the amplification products into the 96-well Elution Plate.
	9.	Method ends. Purified amplification products are eluted into the 96-well Elution Plate sitting in the vacuum manifold. Dispose of the SV 96 Binding Plate after use.


Figure 2.Vacuum manifold disassembly, placement of Elution Plate and reassembly of vacuum manifold for elution of purified amplification products on the Beckman Biomek® 2000.

F. General Guidelines for Adaptation to Alternative Robotic Platforms

This method uses vacuum filtration for binding, washing, and elution of samples. Make sure that the vacuum pump you are using is set to pull a vacuum of 15–20 inches Hg to ensure that sufficient pressure is applied. Vacuum pressure less than 15 inches of Hg may reduce efficiency of recovery and may cause column clogging. Vacuum pressure >20 inches may result in spraying.

An 80% ethanol wash solution is recommended for optimal removal of primers and nucleotides from amplification products larger than 500 bases. For optimal recovery of small amplification products (<500 bases) we recommend increasing the ethanol concentration to 95%.

Pause steps built into the purification procedure improve binding, wash and elution. Removal of these pauses may decrease the purity and recovery of amplification products.

Following Wash #3, drying of the Wizard® SV 96 Binding Plate for at least 4 additional minutes is critical to remove residual ethanol. This drying step may need to be extended for more than 4 minutes to make sure that all residual ethanol is removed. Ethanol contamination in the purified eluate may cause inhibition of downstream reactions.

The recommended elution volume for the SV 96 PCR Clean-Up System is 100µl. This results in approximately 60–70µl of eluate. Decreases in elution volume may increase the concentration of eluted product but may also result in a decrease in the efficiency of recovery.

* 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.