Download Automating the Promega Wizard® SV 96 Plasmid DNA Purification

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