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LIFE SCIENCE ROBOTICS Results High-copy plasmid DNA was purified using the automated Wizard SV 96 Plasmid DNA Purification System method on the MICROLAB STAR workstation. Purity and yield were assessed via absorbance measurements using a Nanodrop* ND-1000 Spectrophotometer (Thermo Fisher, USA). Average yield of high-copy plasmid DNA (phMGFP) from MDS42 E. coli bacteria, based on 24 samples taken at an OD600 of 4, was 8.44 ± 0.93µg with an average concentration of 105.5 ± 11.6 ng/µl. Average purity of the isolated high-copy plasmid, based on 24 samples A260/280 and A260/230 measurements, was 1.92 ± 0.04 and 2.35 ± 0.20, respectively. For cross-contamination evaluation, the average yield of DNA from 8 blank well samples was also measured. Blank sample purity was found to be negligible at 0.01 ± 0.05µg. See Table 1. Assessment Average ± SD Yield 8.44 ± 0.93 µg Concentration 105.5 ± 11.6 ng/µl Purity A260/A280 A260/A230 1.92 ± 0.04 2.35 ± 0.20 Table 1:Summary of results obtained using twenty-four high-copy plasmid DNA samples purified using the automated Wizard SV 96 Plasmid DNA system method on the Hamilton MICROLAB STAR workstation accessed via absorbance measurements using a spectrophotometer (OD600 = 4). Purified plasmid DNA samples were also assessed via automated fluorescent sequencing, restriction enzyme digestion and digital quantitation of bands from agarose gel analysis. ABI Fluorescent BigDye* Terminator sequencing results were obtained from 15 purified plasmid DNA samples with a 600 base read at >98% integrity, demonstrating the quality of the purified product. Restriction enzyme digests using BglII and NcoI resulted in complete digestion of 1µg purified plasmid DNA using 10U/µg enzyme after 1 hour at 37ºC. Finally, percent recovery of supercoiled plasmid DNA for 16 samples was assessed using a fluorescent gel scanner (FMBIO, Hitachi-MiraiBio, USA) and found to be >80% supercoiled DNA as judged by digital quantitation from scans of ethidium bromide stained agarose gels. Recovery and purity data for plasmid DNA samples purified using this automated procedure were also found to correspond with results obtained using the manual purification protocol for this system (3). Refer to Promega TB#272 for details on the manual processing procedure. ® ® By Sarah Shultz, Promega Corporation, and Rick Luedke, Hamilton Robotics Conclusions The automated method described in this application note results in successful isolation and recovery of high-quality plasmid DNA directly from pelleted bacterial culture. Coupled with Hamilton’s MICROLAB STAR Workstation, the Promega Wizard SV 96 Plasmid DNA Purification System represents a high-throughput, easy-to-use approach for reliable recovery of purified DNA fragments suitable for many downstream applications. References (1) W izard SV 96 Plasmid DNA System Technical Bulletin #TB272, Promega Corporation. “http://www.promega.com/tbs/tb272/tb272. html”http://www.promega.com/tbs/tb272/tb272.html (2) A utomated Wizard SV 96 Plasmid DNA Purification System Protocol, EP002, Promega Corporation, “http://www.promega.com/tbs/ep002/ ep002.html”http://www.promega.com/tbs/ep002/ep002.html (3) D onald Smith, Brisco, P., Krueger, S., Stencel, E., Shiels, G., and Grosch, D. (1999) Wizard SV 96 Plasmid DNA Purification System: High Quality Plasmid DNA for Use in Fluorescent Sequencing MethodsThe, Promega Notes 73, Pages 8-11. * All trademarks are the property of their respective owners. Lit. No. L50137 ©Hamilton Company USA 07/09 w w w. h a m i l t o n r o b o t i c s . c o m Automating the Promega Wizard SV 96 Plasmid DNA Purification System on the Hamilton MICROLAB STAR Workstation Hamilton Company 4970 Energy Way Reno, Nevada 89502 USA Toll-Free: 800-648-5950 Telephone: +1-775-858-3000 Fax: +1-775-856-7259 [email protected] Hamilton Bonaduz AG Via Crusch 8 CH-7402 Bonaduz Switzerland Telephone: +41-81-660 60 60 Fax: +41-81-660 60 70 [email protected] Figure 1: The Hamilton MICROLAB® STAR Automated Pipetting Workstation Abstract Plasmid DNA is useful in a wide range of molecular biology applications. However, purification of plasmid DNA from pelleted bacterial culture can be time-consuming when done manually. Automation of this process can deliver significant increases in throughput as well as higher levels of precision and accuracy. We have developed an automated method that enables processing of multiple 96-well plates per day using the Promega Wizard SV 96 Plasmid Purification System on the Hamilton MICROLAB STAR workstation. Here we provide an example of high-copy plasmid DNA purification with this easy-to-follow automated protocol. High-purity plasmid DNA is isolated consistently from bacterial culture samples with no detectable crosscontamination, and can be used directly in downstream sequencing and restriction enzyme digestion. Introduction Plasmid DNA is a versatile and important part of many molecular biology techniques. High quality and purity of the plasmid DNA are critical to ensuring optimal performance in downstream applications. Automation of the purification process can improve consistency while increasing throughput. Here we report on the development of an automated plasmid DNA procedure using the Promega Wizard SV 96 Plasmid Purification System on the Hamilton MICROLAB STAR Workstation. Basic Isolation Procedure The basic steps in plasmid DNA isolation are: disruption of the cellular wall to create a lysate, separation of plasmid DNA from cell debris and genomic DNA, and purification of the plasmid DNA away from other cellular components. When performed manually, plasmid isolation is both labor- and time-intensive. The Promega Wizard 96 Plasmid Purification System is a convenient, filter-based plasmid DNA purification product designed to meet the needs of researchers who want to isolate plasmid DNA more quickly and with less human intervention. Automation of this system on the Hamilton MICROLAB STAR Workstation (Figure 1) delivers a reliable and reproducible method for high-throughput plasmid isolation. Overnight Cultures Centrifuge. Resuspend. Lyse. Neutralize. Lysate Clearing Plate Transfer Lysates Clear Lysates and vacuum-based tasks. The STAR line platform uses air displacement technology, which increases pipetting accuracy and repeatability and eliminates sample contamination or dilution effects commonly associated with fluid-based systems. Hamilton’s TADM (Total Air Displacement Monitoring) technology enables real-time monitoring and recording of pipetting performance for complete process control. The STAR workstation can be configured with multiple arms, each arm housing multiple pipetting channels or labware gripping options. Pipetting channels and labware grippers move independently for increased efficiency and support the use of a wide range of labware. An autoload option provides real-time barcode tracking of samples, labware, racks and carriers as they are loaded onto the deck. Figures 3a & 3b show an example of the MICROLAB STAR Automated Pipetting Workstation deck. All workstation functions and integrated third-party devices are controlled by Hamilton’s VENUS ONE software. Binding Plate Bind DNA Wash Elute DNA Figure 3a: MICROLAB STAR Automated Pipetting Workstation Elution Plate Highly Pure Plasmid DNA Figure 2:Flow diagram of plasmid DNA isolation and purification using the automated protocol. The automated Wizard SV 96 Plasmid DNA Purification System protocol carried out on the Hamilton STAR pipetting workstation described in this application note provides a simple and reliable method for the rapid isolation of plasmid DNA in a silica-membrane, 96-well, high-throughput format. Figure 2 shows a flow diagram of plasmid DNA isolation and purification using this system (1). Up to 96 plasmid samples can be processed in 30 minutes or less. The purified plasmid can then be used directly for automated fluorescent DNA sequencing and other standard molecular biology techniques such as restriction enzyme digestion. The MICROLAB STAR Workstation is a flexible, automated robotic pipetting system that can be adapted to perform various liquid handling Materials and Methods Automated Method System Requirements Part Number MICROLAB STAR Autoloading liquid handling workstation with eight (8) independent pipetting channels, one (1) 96 channel Multi-Probe Head and iSWAP labware manipulating arm Hamilton 173000-036 DWP Carrier (5 SBS positions) Hamilton 182090 MTP Carrier (5 SBS positions) Hamilton 182365 Disposable Tip Carrier (5 racks, 480 tips) Hamilton 182085 Basic Vacuum System (BVS) (include vacuum pump, controller, and collection bottle) Hamilton 190033 Teleshake Shaker for DWP Hamilton 187149 Instrument Controller Hamilton 6436-01 VENUS ONE Software Hamilton 911004-09 Teleshake Shaker Labware Requirements Part Number 300 µl CO-RE disposable filter tips in blue hanging rack Hamilton 235903 1,000 µl CO-RE disposable filter tips in blue hanging rack Hamilton 235905 96-well deep-well pyramid-bottom reservoir plates Hamilton 56669-01 12-column deep-well reservoir plates Promega V6791 Wizard® SV 96 Plasmid DNA Purification System Promega A2250 The following materials are supplied by the user: LB agar plates with appropriate antibiotic 95% ethanol liquid culture medium containing antibiotic Figure 3b: Close-up of deck layout on MICROLAB STAR Workstation The Hamilton MICROLAB STAR Workstation configured for the Wizard SV 96 Plasmid Purification System is shown in Figure 4. tabletop centrifuge capable of 1,500 x g, fitted with 96-well plate adapters Basic Vacuum System DWP Carrier Tip Carrier MTP Carrier Reagent Tub Carrier Figure 4:Deck layout on MICROLAB STAR Workstation showing the labware location at the start of the method. Reagent Dispense Volumes Prior to starting the run, reagents must be manually placed onto the deck of the STAR Workstation. Place four 96-well deep-well pyramid bottom reservoir plates on the deck on the DWP carrier at grid position 9, sites 1 – 4, and one 12-column deep-well reservoir plate at grid position 9, site 5. Fill the reservoirs as follows: Site 1: 40 ml Wizard SV 96 cell resuspension solution Site 2: 40 ml Wizard SV 96 cell lysis solution Site 3: 84 ml Wizard SV 96 neutralization solution Site 4: 220 ml Wizard SV 96 wash solution (ethanol added – refer to the bottle label for instructions) Site 5: (column 1) 13 ml Wizard SV 96 nuclease-free water Method Steps - (2) contains a more detailed description of each method step. Resuspend bacterial cell pellets Cell lysis System pause for cell lysis Neutralization Transfer lysate to clearing plate Vacuum transfer from lysate clearing plate to binding plate Restack filtration stack Bind plasmid DNA to binding plate Neutralize solution wash Wash binding plate Dry binding plate Assemble for elution Elute plasmid DNA from binding plate Elute purified plasmid DNA into the elution plate