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Fully Automated Method for Plasmid Mini-Preps Using Montage Plasmid Miniprep 96 Kits on the Biomek® FX Nucleic Acid Preparation System

Introduction

The accelerated pace of DNA sequencing projects has increased the need for automation of template preparation. Here we describe a fully automated plasmid preparation method using the
MultiScreen₉₆ Separation System on the Beckman Biomek^® FX automated pipettor. The procedure requires no manual intervention as the MultiScreen system employs a vacuum-based methodology.

The protocol was developed and optimized based upon standard methods. Culture and lysis conditions may be optimized for individual needs. The protocol clears the lysate by filtration and relies primarily on vacuum for the bulk of the sample processing. The purified samples are of sufficient quantity and quality for sequencing applications with a processing time of approximately 30 minutes per prep. Due to the large deck area available, the process could be modified to handle multiple manifolds and plates at one time to increase the sample throughput.

Configuration of the Biomek FX Deck Setup

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 37 ºC at 320 r.p.m. for 20–24 hours.

2. Centrifuge at 1500×g 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 150 µL of Solution 1 (P6) and mix for 20 cycles. Wash tips (P10).

5. Add 150 µL of Solution 2 (P9) and mix for 30 cycles. Wash tips (P10).

6. Incubate for 2 minutes.

7. Add 150 µL of Solution 3 (P12) and mix for 20 cycles.

8. Transfer 200 µL 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 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 200 µL of Wash solution (P8) to the MultiScreen96 PLASMID plate (SPE1).

12. Filter at 24 inches Hg for 5 minutes.

13. Add 50 µL of resuspension solution (P11) to the MultiScreen96 Plasmid plate. Incubate for 2 minutes.

14. Mix resuspension solution for 30 cycles and transfer 45 µL of sample to V-bottom collection plate (P7).

Figure 1. 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. Four 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. 1.0 mL cultures were grown in deep well blocks for approx. 22 hours at 37^oC. After growth, samples were pooled to ensure homogeneity throughout the culture and redistributed to four deep well blocks. The blocks were centrifuged to pellet the bacterial cells and the samples were purified on the Biomek FX. 10 µL of the recovered samples were run out on a 1.0% agarose gel. M=Low DNA mass ladder (8 µL, 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:	2.63	2.537	2.759	2.7

Figure 2. Electropherogram of a plasmid template prepared with the Montage Plasmid Miniprep₉₆ Kit. Samples were sequenced using Big Dye^® terminator chemistry (ABI) using 2 µL of sample as template in a 5 µL total reaction volume. Samples were purified using Millipore MultiScreen₃₈₄-SEQ plates, resuspended in 20 µL of 0.3mM EDTA as loading buffer and run on a MegaBace^® 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.

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