eLabNotebook - Promega's Cells to Gels MagneSil* Plasmid Purification Protocol on the Biomek® FX

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MagneSil* Biomek® FX

Promega's Cells to Gels MagneSil* Plasmid Purification Protocol on the Biomek® FX

Promega Corporation – www.promega.com

Before You Begin

A. Preparation of Bacterial Cultures:

Inoculate single-colony isolates from agar plates into a square-well, 2ml culture plate (i.e., Fisher Cat.# 07-200-700) containing the desired volume of media (maximum of 0.3ml). Both Terrific Broth (1.2% tryptone, 2.4% yeast extract, 0.4% glycerol in 0.089M potassium phosphate buffer) and CIRCLEGROW* (BIO 101, Inc. Part# 3000-132) yield high cell mass. However, if consistent mass from well-to-well is your primary goal and high plasmid yield is secondary, you may want to use LB. Cover the plates with aluminium foil and incubate at 37°C with shaking (~800rpm) for 16 to 18 hours.

B. Automated Plasmid Purification on the Biomek® FX Robotic Workstation

Materials to Be Supplied by the User:
• 96-well, U-bottom plates (Greiner Cat.# 650101)
• Innovative Microplate reagent reservoirs (Innovative Microplate Cat.# S30014)
• V-bottom, deep-well culture plates (Fisher Cat.# 07-200-700, Costar Cat.# 3960)
• 80% ethanol
• 100% isopropanol
• 5M NaCl

Many types of reservoir and deep-well culture plates are available. However, the MagnaBot* 96 Magnetic Separation Device is specifically designed for use with Greiner U-bottom microplates. Optional lids are available for Innovative Microplate reservoirs (Innovative Microplate Cat.# A50014)

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

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

Deck Position(s)	Equipment
1/1, 1/2 and Reagents	P250 tip rack assembly
Heat Block	A Heat Transfer Block is placed on top of the Heating/Cooling ALP
A164	Greiner U-bottom plate containing 50µl/well MagneSil* RED
A220	Greiner U-bottom plate containing 50µl/well MagneSil* BLUE
Bind, Clearing and Eluate	Empty Greiner U-bottom plates
A711, A712, A713, A165, ethanol, NaCl, and IPA	Innovative Microplate reservoirs
Culture Plate	96-well culture plate containing 150µl/well overnight bacterial culture is placed on top of the orbital shaker ALP
Harvest	An empty Greiner U-bottom plate is placed on top of a MagnaBot* 96 Magnetic Separation Device fitted with a 3/16 inch spacer

Fill the reservoirs with reagents as follows:

A711	20ml Cell Resuspension Solution
A712	25ml Cell Lysis Solution
A713	25ml Neutralization Solution
A165	20ml Elution Buffer
ethanol	40ml 80% ethanol
IPA	25ml 100% isopropanol
NaCl	20ml 5M NaCl

Once the deck and reagents are set up as shown, turn on the circulating water bath and set to 65°C. This will warm the Heat Block to the desired temperature for drying. The method can now begin. The Biomek® FX method is available from Promega Technical Services. Total run time using the set-up and equipment described is approximately 41 minutes.

Note: Innovative Microplate reservoirs require a dead volume of ~15ml. The excess reagent can be recovered at the end of the procedure.

D. Overview of the Biomek® FX Cells to Gels Method

This is a step-by-step description of the Biomek® FX single-plate method for purifying plasmid DNA. Using this procedure, plasmid yields from 150µl cultures of DH5a[pGEM*-3Zf(+)^(c)], average >1µg. Excellent results were obtained using this purified plasmid as template for Big Dye* terminator sequencing on both the ABI 377 and ABI 3700 instruments. In addition, the isolated plasmid was successfully used as substrate for enzymatic digestion and in PCR applications. Alternate host/vector systems may give different yields, depending on plasmid copy number.

Prior to running this method, please check that the deck of the Biomek® FX has been set according to the initial configuration described in Figure 1. Be sure that the circulating water bath has been turned on and set to 65°C before starting the method.

	1.	Processing begins with the culture plate on the orbital shaker. The first step is cell harvesting.
	2.	MagneSil* BLUE is mixed to homogeneity, and 25µl is added to each well of the culture plate. Mixing is accomplished using the orbital shaker (900rpm for 15 seconds).
	3.	5M NaCl (27µl) is added to each well to a final concentration of 0.9M. The mixing method described in step 2 is repeated.
	4.	Next, 106µl (0.6 volumes) of 100% isopropanol is added to each well. Mixing here must be more vigorous because the mixture tends to be biphasic. The orbital shaker setting is increased to 1,000rpm for 40 seconds. Once thoroughly mixed, the plate is incubated at room temperature for 1 minute.
	5.	The cell harvest mixture is transferred to the empty U-bottom (“Harvest”) plate on top of the MagnaBot* Magnetic Separation Device and a 20-second pause is allowed for separation.
	6.	The supernatant is removed and discarded. The culture plate is removed to a vacant location on the deck and the Harvest plate is returned to the orbital shaker. A new U-bottom (“Clearing”) plate is moved on top of the MagnaBot* 96 Magnetic Separation Device for later clearing of the lysate.
	7.	Cell Resuspension Solution (60µl) is added to each well of the Harvest plate and the MagneSil*-bound cells are resuspended using the orbital shaker (1,400rpm for 2 minutes).
	8.	Cell Lysis Solution (80µl) is added to each well of the Harvest plate and mixed using the orbital shaker at 700rpm for 3 minutes.
	9.	MagneSil* RED is mixed to homogeneity during Step 7. Thirty-five microliters of MagneSil* RED are then delivered to each well of the “Bind” plate.
	10.	Neutralization Solution (80µl) is added to each well of the Harvest plate and mixed by pipetting a volume of 150µl up and down five times. The lysate is then transferred to the Clearing plate on top of the the MagnaBot* Magnetic Separation Device. Thirty seconds are allowed for clearing of debris. The Harvest plate is then moved off the orbital shaker to a vacant location on the deck.
	11.	Cleared lysate (20µl) is transferred to the Bind plate containing 35µl MagneSil* RED. Mixing is accomplished by pipetting up and down 30 times. At this Step, the plasmid DNA binds to the MagneSil* RED.
	12.	The Clearing plate (containing MagneSil* BLUE) is removed and the Bind plate is placed on top of the MagnaBot* Magnetic Separation Device. After a 20-second pause for separation, spent lysate is removed and discarded.
	13.	The Bind plate is placed on the orbital shaker. MagneSil-bound plasmid is washed by adding 100µl of 80% ethanol and resuspending with the orbital shaker at 800rpm for 60 seconds. The plate is then moved to the MagnaBot Magnetic Separation Device and the wash solution is removed and discarded. This Step is repeated two additional times.
	14.	The Bind plate is moved to the Heat Block for five minutes to remove residual ethanol. 15. The Bind plate is returned to the orbital shaker and 50µl of Elution Buffer is added to each well. DNA is resuspended by shaking at 1,200rpm for 90 seconds. A 1-minute pause is then allowed.
	16.	The plate is moved onto the the MagnaBot* Magnetic Separation Device for final separation. An on-magnet mix ensures that any carryover of MagneSil* is avoided. The purified plasmid eluates are transferred to the “Eluate” plate.

E. General Guidelines for Cells To Gels Plasmid Purification on Alternate Robotic Platforms Although the Cells to Gels plasmid purification protocol was developed using the Biomek® FX robotic workstation, the method is amenable to alternate robotic platforms. Some general guidelines are presented below.
	A. Cell Harvesting

Table 1.Volumes of Reagents Required for Cell Harvesting.

Culture Volume	5M NaCl	MagneSil* BLUE (A220D/A220F)	100% Isopropanol	Total Volume
150µl	27µl	25µl	106µl	308µl
200µl	36µl	25µl	142µl	403µl
250µl	45µl	25µl	177µl	497µl
300µl	54µl	25µl	212µl	591µl

	1.	Add 25µl of MagneSil* BLUE to each well of the culture plate and mix to homogeneity either with disposable tips (8–10 mixes) or on an orbital shaker at 600–800rpm for 20 seconds.
	2.	Add 5M NaCl (user supplied) to a final concentration of 0.9M in the culture plate using the volumes shown in Table 1. The mixing method described in Step 1 is repeated.
	3.	Next, add 0.6 volumes of 100% isopropanol (user supplied) to each well of the culture plate using the volumes shown in Table 1. At this step, mixing is more vigorous as the mixture tends to be biphasic. Once thoroughly mixed, incubate at room temperature for 1 minute.
	4.	From this point, all processing steps are performed using 300µl roundbottom microplates (Greiner, Part #650101). The cell harvesting mixture is pipetted into a plate positioned on a MagnaBot* Magnetic Separation Device fitted with a 3/16 inch Spacer. The MagneSil*-bound cells will separate in approximately 20 seconds. Supernatants may appear somewhat cloudy or translucent. This cloudiness is caused by the precipitation of proteins and media components after isopropanol addition. The supernatant is withdrawn and discarded. Transfer, separation, and supernatant disposal may need to be repeated several times, depending on your starting culture volume.

B. Plasmid Purification

	1.	Cell Resuspension Solution (60µl) is added to the Harvest plate to disperse the MagneSil* pellets. While it is possible to resuspend these pellets with disposable tips, the procedure works best on an orbital shaker. The shaker speed must be determined empirically to ensure uniform resuspension while avoiding spillage. A good starting point is 900–1,000rpm.
	2.	Cell Lysis Solution (60µl) is added, shaking gently for 3 minutes (600–700rpm).
	3.	Neutralization Solution (80µl) is added. Volume restrictions for the microplate require that disposable tips are used for mixing the lysate to uniformity. Pipetting 170µl up and down 5–6 times is usually sufficient.
	4.	The lysate is transferred to a fresh Clearing plate positioned on top of the MagnaBot* Magnetic Separation Device. The MagneSil* BLUE added for cell harvesting now serves the purpose of removing bound debris. This clearing step may take up to a minute, depending on the amount of cell mass processed. Some MagneSil*-bound debris will adhere to the plate and be left behind. This does not affect the overall yield and, in fact, speeds the lysate clearing process.
	5.	Approximately 180–200µl of cleared lysate will result. This is transferred to a third Greiner plate containing 35µl/well of MagneSil* RED binding resin. Mixing at this step is accomplished by pipetting 180µl up and down 20 times with disposable tips. This is required due to the high volume in the microplate. Use a pipetting speed just fast enough to keep the MagneSil* RED uniformly dispersed. Over-mixing or mixing at high speed will cause nicking of the plasmid over time. This is not a problem for sequencing applications, but is critical if you need to purify a high percentage of supercoiled plasmid.
	6.	The binding plate is then moved to the MagnaBot* Magnetic Separation Device. After a 20–30 second pause, separation occurs. This separation should be much faster than that seen for lysate clearing. The supernatant is removed and discarded.You may wish to keep this supernatant when performing the procedure of the first time to check the efficiency of binding (Step 5). High salt is already present, so precipitation of any remaining plasmid can be accomplished by simply adding 2 volumes of 95% ethanol and centrifuging ten minutes at 10,000 × g.
	7.	The MagneSil-bound plasmid is washed by adding 100µl of 80% ethanol and resuspending using an orbital shaker set at 800–900rpm for 60 seconds. The shaker speed must be determined experimentally to ensure uniform resuspension while avoiding spillage. After shaking, the plate is moved to the MagnaBot Magnetic Separation Device for separation and waste removal. This Step is repeated two additional times.
	8.	The MagneSil* pellets must be dried to remove residual ethanol. Although this can be accomplished by air-drying for 15 minutes, best results are obtained by moving the plate to a 65°C bench-top heater fitted with a Heat Transfer Block (Cat.# Z3271) for five minutes. Use of this block lowers the ethanol concentration in the eluted plasmid from 3.5% to 0.5%. This dramatically improves signal strength in sequencing applications using the purified plasmid as template. Using this protocol, a 7.5-minute drying step will reduce ethanol contamination to 0.5% or less.
	9.	To elute the plasmid, the MagneSil* particles are resuspended in Elution Buffer. A minimum volume of 50µl is required for resuspension using an orbital shaker at 1,000–1,200rpm. As much as 100µl can be used for high copy plasmids, but high volume is not required for efficient elution.

* 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.