eLabNotebook - Promega's Automated SV 96 Total RNA Isolation System on the Biomek® FX

eLabNotebook > Nucleic Acid Prep & Purification > RNA Purification > Promega SV 96 Total RNA
Isolation Biomek® FX

Promega's Automated SV 96 Total RNA Isolation System on the Biomek® FX

Promega Corporation – www.promega.com

Before You Begin

Materials to Be Supplied by the User:

• ethanol, 95%, RNase-free (120ml per 96-well plate)
• 10X phosphate-buffered saline (PBS), sterile (for cultured cells)
• vacuum pump capable of 15–20 inches of Hg (e.g., Fisher Cat.# 01-092-29)
• vacuum trap for waste collection
• vacuum tubing (e.g., neoprene tubing, Fisher Cat.# 14-171B)
• for Biomek® FX only: Pyramid Bottom Reservoir Plates (2; Innovative Microplate, Cat.# S30014)

A. Preparation of Solutions

Prior to beginning the procedure with a new SV 96 Total RNA Isolation System, dilute the provided solutions as follows:

SV RNA Lysis Buffer: Add 1ml ß-mercaptoethanol (BME) to 50ml SV RNA Lysis Buffer. After adding BME, mark on the bottle that this step has been performed. Store the SV RNA Lysis Buffer with BME at 4°C.

DNase I: Add 312.5µl of Nuclease-Free Water (supplied) to the lyophilized DNase I. Gently mix by swirling. Do not vortex. One vial is sufficient for one 96-well plate. If processing less than a whole plate, we recommend dividing the rehydrated DNase into working aliquots using sterile, RNase-free microcentrifuge tubes. Each RNA purification requires 2.5µl of rehydrated DNase I. Store the rehydrated DNase I at –20°C.

SV RNA Wash Solution: Add 100ml of 95% ethanol to the bottle containing 58.8ml concentrated SV RNA Wash Solution. After adding ethanol, mark on the bottle that this step has been performed. The SV RNA Wash Solution is stable at 22–24°C when tightly capped.

SV DNase Stop Solution: Add 20ml of 95% ethanol to the bottle containing 13.25ml concentrated SV DNase Stop Solution. After adding ethanol, mark on the bottle that this step has been performed. The SV DNase Stop Solution is stable at 22–25°C when tightly capped.

B. Sample Preparation Before Automated Processing

Before placing the plate containing cells on the deck of the robot, wash the cells once with 1X PBS. Make sure to remove media or PBS before placing cells on the deck of the robot for processing. SV RNA Lysis Buffer should be added to cells alone.

C. Intial Deck Layout for the Beckman Biomek® FX Workstation

(Click to Enlarge)

ALP Name	Part Siting on ALP
Tip Loader	200µl non-ART Biomek® FX tips
P1	200µl ART Biomek® FX tips
P2	200µl non-ART Biomek® FX tips
P3	200µl ART Biomek® FX tips
P4	96-well, flat-bottom sample plate
P5	Pyramid bottom reservoir plate containing 40ml SV RNA Lysis Buffer (BME added)
P7	Swap spot
P8	Greiner 96-well, round-bottom plate containing 31µl of DNase Solution per well
P9	Greiner 96-well, round-bottom plate containing 125µl nuclease-free water per well
P11	Pyramid bottom reservoir plate containing 130ml SV Wash Solution (ethanol added)
P12	Greiner 96-well, round-bottom plate containing 225µl DNase Stop Solution per well
P13	96-well, flat-bottom elution plate
SPE ALP	SPE ALP: Vacuum filtration manifold base, elution spacer, 36mm collar, SV 96 Binding Plate

D. Pre-Run Beckman Biomek® FX Specific Recommendations

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

Prior to the first run of the SV96 Total RNA Purification method on the Beckman Biomek® FX, check all Gripper moves to ensure that the vacuum manifold disassembly and reassembly for elution is correct. Our experience indicates that proper configuration of the Gripper moves is essential to ensure the success of SV 96 methods on the Biomek® FX. Not performing the Gripper test evaluation may result in failure of vacuum manifold disassembly and reassembly and may damage your Biomek® FX instrument.

Follow the instructions provided: Evaluation of Biomek® FX SV 96 Purification 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 method: “BFXSV96griptest”. This method can be obtained by contacting Promega.

E. Description of Automated SV 96 Total RNA Isolation

This overview describes the general liquid handling steps required for automated SV 96 Total RNA Isolation and can be adapted to a variety of automated liquid-handling robots. For additional information for adaptation to liquid-handling robots other than those referenced above, please see Section VII, General Guidelines for Adaptation to Alternative Robotic Platformsi>

	1.	Cell Lysis. One hundred microliters of SV RNA Lysis Buffer is transfered from a reservoir to the 96-well, flat-bottom sample plate containing cells alone. The contents are mixed with pipet tips to completely lyse cells.
	2.	Transfer Cell Lysates. The cell lysate contained in the 96-well, flat-bottom sample plate is transfered to the SV 96 Binding Plate sitting on top of the vacuum manifold apparatus.
	3.	Bind Total RNA to SV 96 Binding Plate. Once all the cell lysate has been transferred to the SV 96 Binding Plate, the vacuum is applied, and cell lysate is pulled through the SV 96 Binding Plate by vacuum for one minute. During this vacuum, total RNA binds to the SV 96 Binding Plate.
	4.	Wash #1 SV 96 Binding Plate. Five hundred microliters of SV RNA Wash Solution is dispensed to each well of the SV 96 Binding Plate.Vacuum is applied, and the Wash Solution is drawn through the SV 96 Binding Plate by vacuum for one minute.
	5.	DNase Treat Samples. Twenty five microliters of prepared DNase Solution is transfered from the reservoir to each well of the SV 96 Binding Plate. The robot pauses for 10 minutes to allow for DNase incubation. DNase incubation is typically 10 minutes, but can be lengthened to 20 minutes.We do not recommend greater than a 20-minute DNase incubation.
	6.	DNase Stop Solution. The DNase is inactivated by adding 200µl of DNase Stop Solution from the reservoir to each well of the SV 96 Binding Plate. Vacuum is applied, and the DNase Stop Solution is drawn through the SV 96 Binding Plate by vacuum for 30 seconds.
	7.	Wash #2 SV 96 Binding Plate. Five hundred microliters of SV RNA Wash Solution is dispensed to each well of the SV 96 Binding Plate. Switch the vacuum on, and the SV RNA Wash Solution is pulled through the SV 96 Binding Plate by vacuum for one minute.
	8.	Drying/Removal of Residual Alcohol. The vacuum remains on for three more minutes to remove any residual ethanol from the SV 96 Binding Plate.
	9.	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 manifold collar from the vacuum manifold to holding position. The Gripper then moves the 96-well, flat-bottom elution plate into the vacuum manifold and reassembles the vacuum manifold stack by moving the SV 96 Binding Plate and manifold collar back onto the vacuum manifold.
	10.	Elution of Purified Total RNA. One hundred microliters of nuclease-free water is transfered from the reservoir to each well of the SV 96 Binding Plate. Incubate at room temperatue for 1 minutes. Vacuum is applied, and the Nuclease-Free Water is pulled through the SV 96 Binding Plate, eluting the total RNA into the 96-well, flat-bottom plate.
	11.	Method Ends. Purified total RNA has been eluted into the 96-well, flat-bottom plate that is sitting in the vacuum manifold position A6. Dispose of the Binding Plate after use.

General Guidelines for Adaptation to Alternative Robotic Platforms

Because the SV 96 Total RNA Isolation System is used to isolate RNA from tissue culture cells and tissue lysates, we recommend using aerosol-resistant tips for this method to decrease the chance of contaminating samples with RNases. If your robotic platform uses fixed tips, be sure that the tips are washed thoroughly between pipetting steps. Also, if system liquid is used to perform pipetting steps, be sure to limit the exposure of samples to system liquid (a potential source of RNase contamination) during all pipetting steps by increasing the volume of leading air gaps that are used for pipetting.

This method uses vacuum filtration of samples for binding, washing, and elution. Make sure that the vacuum pump you are using is set to pull a vacuum of 15–20 inches Hg to ensure that a sufficient vacuum pressure is being used. Vacuum pressure less than 15 inches of Hg will result in reduced purified total RNA yield and purity and may cause column clogging when processing tissue lysates.

DNase I Solution volumes provided in the SV 96 RNA Isolation Kit are limiting. Therefore, use exactly the recommended volumes described in this Automated Technical Bulletin. When performing the DNase I treatment of samples on the membrane of the SV 96 Binding Plate, be sure to pause 10 minutes to degrade genomic DNA before adding the SV DNase Stop Solution.

Following Wash #2, drying the Wizard* SV 96 Binding Plate for at least 3 additional minutes is critical to remove residual ethanol. This drying step may need to be extended for more than 3 minutes to make sure that all residual ethanol is removed. Ethanol contamination in the RNA eluate can inhibit downstream reactions such as RT-PCR.

The recommended elution volume for the SV 96 Total RNA Isolation System is 100µl resulting in approximately 60–70µl of eluted RNA. Decreases in elution volume will result in concomitant decreases in the volume of eluted material and RNA yield.

* 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.