eLabNotebook - Millipore's Fully Automated Method for Plasmid Mini-Preps Using Montage* Plasmid Miniprep96 Kits on the Biomek® FX

eLabNotebook > Nucleic Acid Prep & Purification > Plasmid DNA Purification > Millipore Montáge*
Plasmid Miniprep 96 Biomek® FX

Millipore's Fully Automated Method for Plasmid Mini-Preps Using Montáge* Plasmid Miniprep96 Kits on the Biomek® FX Nucleic Acid Preparation System

Millipore– www.millipore.com

Figure 1. Configuration of the Biomek FX Deck Setup

(Click to Enlarge)

Prior to starting:
Make sure deck is set up as follows:

P1	P200 Tips for alkaline lysis
P2	P200 Tips for resuspension
P4	Deep well block with pelleted cells
P6	Reservoir w/Solution 1
SPE1	PLASMID plate in Millipore manifold
P7	V-bottom storage plate
P8	Reservoir w/Wash solution
P9	Reservoir w/Solution 2
P10	Reservoir w/water for tip washing
P11	Reservoir w/Resuspension solution
P12	Reservoir w/Solution 3
Holder w/framing tool: Millipore collar with Lysate Clearing plate on top.

Procedure for Partial Lysate Protocol

	1.	Inoculate E. coli into 1 ml aliquots of 2X LB plus appropriate antibiotic in sterile 96 deep well blocks. Cover plates and secure in incubator. Incubate at 37ºC at 320 r.p.m. for 20-24 hours.
	2.	Centrifuge at 1500xg for 5 minutes. Decant culture supernatant to a container for proper disposal.
	3.	Place deep well block with pellets on the Biomek FX in P4. Start program.
	4.	Add 150µl of Solution 1 (P6) and mix for 20 cycles. Wash tips (P10).
	5.	Add 150µl of Solution 2 (P9) and mix for 30 cycles. Wash tips (P10).
	6.	Incubate for 2 minutes.
	7.	Add 150µl of Solution 3 (P12) and mix for 20 cycles.
	8.	Transfer 200µl of the lysate from the deep-well plate (P4) into the Lysate Clearing plate (Holder). Unload tips.
	9.	Gripper moves the Lysate Clearing plate and collar (Holder) to vacuum manifold (SPE1). Filter for 3.0 minutes at 24 inches Hg and collect cleared lysate into the PLASMID plate.
	10.	Discard the Lysate Clearing plate. Gripper moves the PLASMID plate to the top of the vacuum manifold. Filter at 24 inches Hg for 8 minutes or until all wells are empty.
	11.	Load tips and add 200µl of Wash solution (P8) to the MultiScreen96 PLASMID plate (SPE1).
	12.	Filter at 24 inches Hg for 5 minutes.
	13.	Add 50µl of resuspension solution (P11) to the MultiScreen96 PLASMID plate. Incubate for 2 minutes.
	14.	Mix resuspension solution for 30 cycles and transfer 45µl of sample to V-bottom collection plate (P7).

Figure 2. Plasmid DNA purified with the Montage kit on the Biomek FX

Plasmid minipreps were performed using the Montage Plasmid Miniprep96 Cleanup Kit on the Biomek FX automated pipettor. 4 plates were processed according to the procedure outlined above. pLH2 plasmid shown above is a pUC19 plasmid with a 2.0Kb insert from a HindIII digested ? phage. 1.0ml cultures were grown in deep well blocks for approx. 22 hours at 37oC. After growth, samples were pooled to ensure homogeneity throughout the culture and redistributed to 4 deep well blocks. The blocks were centrifuged to pellet the bacterial cells and the samples were purified on the Biomek FX. 10µl of the recovered samples were run out on a 1.0% agarose gel. M=Low DNA mass ladder (8µl, Gibco) are loaded between the sample sets 1-4.

Table 1. Yields for Partial Lysate Plasmid Preps done on Biomek FX

	Prep #1	Prep #2	Prep #3	Prep #4
Average:	2.025	2.074	2.172	2.107
Std Dev:	0.272	0.352	0.247	0.275
CV:	13.44%	16.98%	11.36%	13.03%
Min:	0.849	0.212	1.178	0.968
Max:	0.849	2.537	2.759	2.7

Plasmid preps were performed using the Millipore Montage Plasmid Miniprep96 Kit on the Biomek FX pipettor. 4 plates were processed according to the procedure outlined above. Yields were determined by OD260 readings on a SPECTRAmax PLUS plate reader (Molecular Dynamics). Plate averages, standard deviation and coefficient of variation is noted for each plate as well as the minimum and maximum yield on a particular plate.

Figure 3. Electropherogram of sequence using a plasmid template prepared using the Montage Kits on the Biomek FX

(Click to Enlarge)

Figure 3. Electropherogram of a plasmid template prepared with the Montage Plasmid Miniprep96 Kit. Samples were sequenced using Big Dye terminator chemistry (ABI) using 2µl of sample as template in a 5µl total reaction volume. Samples were purified using Millipore MultiScreen384-SEQ plates, resuspended in 20µl of 0.3mM EDTA as loading buffer and run on a MegaBaceTM DNA Analysis System (Injection: 2kV 100sec. Run: 7kV 150min).

Conclusion: Using the Millipore Montage Plasmid Miniprep96 kit results in highly purified plasmid DNA which can be used in downstream applications such as sequencing or transfections. The DNA yields under these conditions ranged from 2-3 µg per well with CV's under 15% per plate. The protocol is simple, fast, and automation compatible.

* 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.