eLabNotebook - Promega's Wizard MagneSil Tfx* System on the Biomek® FX

eLabNotebook > Nucleic Acid Prep & Purification > Plasmid DNA Purification > Promega MagneSil
Tfx* Biomek® FX

Promega's Wizard MagneSil Tfx* System on the Biomek® FX

Promega Corporation – www.promega.com

Before You Begin

To process one plate of 96 samples:

• Deep-well (2ml) 96-well plate (e.g., Beckman deep-well microtiter plate, #140504)

• 3 boxes of 96 disposable P250 tips (Beckman Cat.# 717251)

• 6 × 96-well plates (Greiner Cat.# 650101 or Promega Cat.# A9161)

• 50ml of 80% ethanol

• 25ml of isopropanol

Note: If deep-well plates other than the Beckman plates are used, pipetting settings for the deep-well stations will have to be optimized.

(Click to Enlarge)
Figure 1. Diagram of the deck layout.

A. Protocols

1. Preparation of Cell Pellets

	Bacterial cells should contain high-copy-number plasmids. For information on selecting and growing bacterial strains, see Sections VII.B–C. As much as 6.0 O.D.600 of total cell mass may be processed per plate well.
	A.	Grow bacterial cells overnight at 37°C in up to 1ml of culture volume in a 2ml deep-well culture plate with square wells (e.g., Beckman deep-well titer plate, item# 140504).
	B.	Pellet cells by centrifuging for 15 minutes at 1,500 × g in a tabletop centrifuge. Pour off the supernatant and blot the plate upside down on a paper towel to remove excess liquid.
	C.	Samples can be processed immediately or covered and stored at –20°C for several weeks. If using a frozen plate, make sure the pellets warm to room temperature before beginning the protocol.

2. Robotic Set Up

	Follow these instructions to prepare plates and reservoirs for the robot deck.
	A.	Add 25ml of isopropanol to the 4/40 Wash Solution bottle and mix by shaking.
Shake the MagneSil* BLUE and RED bottles until the resin is resuspended.
	B.	Add the following solutions to a 96-well Greiner plate, one plate per solution: • 1 plate with 50µl/well MagneSil* BLUE (“Mag BLUE” plate, Figure 1) • 1 plate with 50µl/well MagneSil* RED (“Mag RED” plate, Figure 1) • 1 plate with 25µl/well Endotoxin Removal Resin (“ET Removal Resin” plate, Figure 1) • 1 plate with 125µl/well 4/40 Wash Solution (“4/40 wash” plate, Figure 1) • 1 plate with 100µl/well Nuclease-Free Water (“Elute Plate”, Figure 1)
	C.	Add the following solutions directly to the reservoirs: • 25ml Cell Resuspension Buffer> • 25ml Cell Lysis Buffer> • 25ml Cell Neutralization Buffer> • 50ml 80% Ethanol
	D.	Set the heating ALP to 55–65°C.
	E.	Place the 96-well Greiner plates and reservoirs on the robot deck in the positions indicated in Figure 1 or as suggested in the electronic protocol under Set Up.

3. Robotic Protocol


	A.	Add 90µl of Cell Resuspension Buffer to each plate well. Resuspend the bacterial pellets by pipetting and shaking.
		Note: Cell resuspension in Section C, Step 1, is very important. If the pellets are not completely resuspended, plasmid quality and yield can be affected. If biomass is too high, cell resuspension may be incomplete. During trial runs, stop the protocol after the resuspension step and check for resuspension. If lysis is incomplete, increase shaking time.
	B.	Add 120µl of Cell Lysis Buffer to each well and mix by shaking. Complete lysis is important for yield and quality. Perform trial runs, checking for the completeness of lysis. If lysis is incomplete, decrease the amount of biomass.
	C.	Add 120µl of Cell Neutralization Buffer. Mix the lysate by pipetting and shaking.
	D.	Add 25µl of MagneSil* BLUE. Mix the lysate by pipetting and shaking.
	E.	Transfer the lysate to a new 96-well Greiner plate and place the plate on the MagnaBot* 96. Allow the lysate to clear. A tight pellet should form in the corner of each well, which allows a pipette tip to descend to the center of the well without disturbing the pellet.
	F.	Transfer the cleared lysate to the plate containing Endotoxin Removal Resin and mix by pipetting. The resin and lysate are mixed by pipetting and not by shaking because volumes are too large for shaking.
		Note: There should be no negative effects if a small amount of resin and debris is transferred to the endotoxin removal plate.
	G.	Transfer the plate containing the lysate and Endotoxin Removal Resin to the MagnaBot* 96 and allow the lysate to clear.
	H.	Transfer the cleared lysate to the plate containing MagneSil* RED and mix by pipetting.
	I.	Transfer the plate containing the lysate and MagneSil* RED to the MagnaBot® 96 and allow the lysate to clear. Remove and discard the lysate.
	J.	Remove the plate containing MagneSil* RED from the magnet. Add 100µl of 4/40 Wash Solution and mix by pipetting.
	K.	Transfer the plate to the MagnaBot* 96 and allow the solution to clear, then discard the 4/40 Wash Solution.
	L.	Remove the plate containing MagneSil* RED from the magnet. Add 100µl of 80% ethanol to each well. Mix by shaking.
	M.	Transfer the plate to the MagnaBot* 96 and allow the solution to clear, then discard the 80% ethanol wash.
	N.	Repeat the ethanol step 2 times, for a total of 3 ethanol washes. It is important to remove as much ethanol as possible after the final wash.
	O.	Transfer the plate containing MagneSil* RED to the heating block for 7.5 minutes.
		Note: If a heating/cooling ALP is unavailable, the plate can be dried at room temperature for 10 minutes or longer.
	P.	Remove the plate from the heating block, add 100µl of Nuclease-Free Water, then mix by pipetting.
	Q.	RTransfer the plate to the MagnaBot* 96 and allow the solution to clear. Remove the Nuclease-Free Water containing the plasmid and transfer it to a final elution plate. If small amounts of resin are carried over to the elution plate, remove them by placing the plate on the MagnaBot* 96 for 5 minutes and then transferring the eluate to a new plate.
		Avoid contaminating the Nuclease-Free Water, as biological contaminants will have serious negative effects on transfection.

* 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.