Beckman Coulter, Inc.
Biomedical Research
4300 N. Harbor Blvd.
Fullerton, CA, 92834
Website: http://www.beckmancoulter.com
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High-Throughput Genomic DNA Isolation From Cell Cultures And Mouse Tails
by Hobert Wai, Ph.D., Matthew Cu, Dana P. Campbell, Keith Roby, Terri Grunst and Daniel Kephart
Figure 1A: Deck setup for genomic DNA purification from one 96-well tissue culture plate. The lids from the
tip boxes were used to hold the SV Lysis Buffer, Wash Solution and Nuclease-free water for this method.
Figure 1B: Deck setup for genomic DNA purification from two 96-well tissue culture plates. The lysis buffer, wash solution and nuclease-free water were stored in
96-well pyramid-bottom reservoirs due to high-volume usage. The pipette tips for samples 1 and 2 were kept separate to limit cross contamination.
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Introduction
The ability to isolate pure genomic DNA has allowed scientists to extract and collect important genetic information from various samples. The data obtained from genomic DNA for genotyping analysis, through the years, has proven critical for projects in biomedical research, forensics, and pharmacogenomics. However, conventional procedures for obtaining genomic DNA are labor-intensive and time-consuming. In addition, manual preparation has been susceptible to contamination from different sources. To address these concerns, multiple automated methods have been developed for the isolation of genomic DNA from tissue cell cultures and mouse tail clippings using the Promega Wizard® SV 96 Genomic Purification System (Promega, Inc., Madison Wis.) and Beckman Coulter’s Biomek FX Liquid Handling System (Beckman Coulter, Inc., Fullerton, Calif.). By using a 96-well vacuum filtration procedure to isolate and purify genomic DNA from tissue cell cultures and mouse tail lysates, the need for centrifugation has been eliminated. Washing the bound genomic DNA requires no disassembly of the vacuum manifold and filtrate waste products are delivered directly to a vacuum trap. Genomic DNA is eluted into a convenient 96-well deep-well plate for convenient downstream sample analysis, making the process simple and easy to implement in the genomic research laboratory. The tissue cell culture method is fully automated and capable of isolating and purifying 96 or 192 high quality genomic DNA samples in, respectively, about 14 and 25 minutes. The mouse tail method is also fully automated and can isolate and purify 96 high-quality genomic DNA samples in approximately 27 minutes. The purified genomic DNA has been evaluated for quality, reproducibility, degradation and cross-contamination using spectrophotometry, agarose gel electrophoresis and PCR.
Materials and methods
Reagents
Genomic DNA was isolated and purified from HeLa cells and mouse tail clippings using Promega’s Wizard SV 96 Genomic DNA Purification System. The single-plate kit includes: 50 ml Nuclei Lysis Solution; 30 ml 0.5 M EDTA, pH 8.0; 50 ml Wizard SV Lysis Buffer; 185 ml Wizard SV Wash Solution (concentrated); 1 ml RNase A Solution; 4 mg/ml 150 ml nuclease-free water; 1 Wizard SV 96 Binding Plate; and 1 96-well deep well plate. Reagents required but not supplied in the kit include: 95% Ethanol (made from absolute ethanol — Aaper Alcohol and Chemical Co., Shelbyville, Ky.), 103 phosphate-buffered saline (Invitrogen Corp., Carlsbad, Calif.) to wash tissue culture cells, and Proteinase K (20 mg/ml — Promega Corp.).
Labware
The Promega single-plate kit includes a filter plate and a 96-well deep-well elution plate. Labware required for running the one-plate tissue cell culture method but not supplied in the kit includes: one 96-well tissue culture plate (Becton, Dickinson and Co., Franklin Lakes, N.J.) and three P250 tip boxes (Beckman Coulter, Inc.). For the two-plate tissue cell culture method, the following are required: two 96-well tissue culture plates (Becton, Dickinson and Co.), six P250 tip boxes (Beckman Coulter, Inc.) and three 96-well pyramid-bottom reservoirs (Innovative Microplate, Chicopee, Mass.). Labware required for running the mouse tail method but not supplied in the kit includes: one 96-well deep well plate (Corning, Inc., Life Sciences, Acton, Mass.) for proteinase K tissue digestion, three P250 tip boxes (Beckman Coulter, Inc.) and one 96-well pyramid-bottom reservoir (Innovative Microplate).
Tissue culture and mouse tail sample preparation
A HeLa cell line (ATCC, Manassas, Va.) was grown according to ATCC guidelines. HeLa cells were seeded at a concentration of 1 3 105 cells per well of a 96-well tissue culture plate for isolation of genomic DNA. Prior to running the method, the supernatant was carefully aspirated to prevent loss of any cells. Next, 200 ml of 13 PBS was added to each well to wash the cells. The 1X PBS wash was carefully aspirated and genomic DNA was purified from the cell plate. Purified genomic DNA samples were analyzed by spectrophotometry, PCR amplification and agarose gel electrophoresis of amplified products.
For mouse tails, genomic DNA was isolated from approximately 1.2 cm or 20 mg of mouse tail clippings digested overnight at 55 C with Proteinase K (see Promega Technical Bulletin #303 for preparation of mouse tail lysates). Frozen mouse tail Proteinase K-treated lysates were incubated in a 55 C water bath for 1 hour prior to running the method to prevent clogging of the SV 96 Binding Plate during processing. Purified genomic DNA samples were analyzed by spectrophotometry, PCR amplification and agarose gel electrophoresis of amplified products.
Figure 2: Initial configuration for genomic DNA purification from a single 96-well plate of digested mouse tail. The lids from the tip boxes were used to hold lysis buffer and nuclease-free water. However, wash solution was stored in a 96-well pyramid-bottom reservoir due to high-volume requirement. The circled 96-tip wash, Magbead and Orbital shaker ALPs were not required for this method.
Figure 3: Cross-contamination plate.
Figure 4: Figure 4. PCR analysis of products using b-actin primers and 1 ml of genomic DNA followed by electrophoretic separation on a 2% pre-cast agarose E-Gel. Lanes 1 to 6 and 8 to 12 are 5 ml of b-actin PCR of genomic DNA samples prepared from wells containing HeLa cells separated at 70 V for 30 minutes. Lanes 14 to 22 are 10 ml of b-actin PCR of samples from wells containing no cells separated at 70 V for 30 minutes. A 1-kb DNA size ladder was loaded in lanes 7 and 13 for size determination.
Figure 5: Cross-contamination assessment for mouse tail. Agarose gel electrophoresis pictures of 1.2-kb PCR products using IL1-b primers and mouse tail gDNA purified on the Biomek FX. Lanes 1, 3, 5, 8, 10, and 12 are 10 ml of IL1-b PCR of genomic DNA samples prepared from wells containing mouse tail lysates separated at 70 V for 30 minutes. Lanes 2, 4, 6, 9,and 11 are 10 ml of IL1-bPCR of genomic DNA samples prepared from wells containing blank media. A 1-kb DNA size ladder was loaded in lane 7 for size determination.
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PCR amplification using b-actin and IL1-b
Genomic DNA samples isolated from HeLa and mouse tails were used to generate PCR products using the following protocol: 1 ml of unnormalized purified genomic DNA was amplified in a 25 ml reaction volume using PCR Master Mix (Promega Corp.). Tissue culture genomic DNA was amplified using b-actin primer sets while mouse tail genomic DNA was amplified using IL1-b primer sets. The thermocycling conditions for b-actin amplification were 95 C for 3 minutes, followed by 40 cycles of 95 C for 30 seconds; 55 C, 30 seconds; 72 C, 5 minutes, then a final extension at 72 C for 5 minutes. Thermocycling conditions for IL1-b amplification were: 95 C for 3 minutes, followed by 40 cycles of 95 C for 30 seconds, 55 C for 1 minute, 72 C for 1 minute, then a final extension at 72 C for 5 minutes. An amplified PCR sample of 10 ml was separated by electrophoresis on a 2% pre-cast agarose E-Gel (Invitrogen, Corp.) at 70 V for 30 minutes.
Software and hardware requirements
Biomek FX Liquid Handling System, hybrid version (96-multichannel and Span-8 pods), with version 2.2 software. Equipment required for the Solid-Phase Extraction (SPE) was: Vacuum manifold, 65 mm collar, SPE ALP and SPE ALP Collar.
Biomek FX SV 96 genomic DNA purification methods
Two separate methods for genomic DNA purification from tissue culture cells or mouse tail tissue lysate were written. Figures 1A and 1B show the initial deck layout for the cell culture method to purify one and two tissue culture plates, respectively. Figure 2 shows the initial deck layout for the mouse tail method for one plate of tail clippings. The user has the ability to select either one- or two-plate genomic DNA isolation and purification within the cell culture method by typing in the appropriate number of plates to be prepared. All methods used the 96-multichannel pipetting head exclusively; therefore, 96 samples were processed in parallel. Both the tissue culture cells and mouse tail lysate methods use three boxes of tips for isolation and purification of genomic DNA per 96 samples. Separate boxes of tips are used for each of the reagents: lysis buffer and sample, wash buffer and nuclease-free water. No user intervention is required during processing as the integrated gripper in the multichannel pod handles all labware movements including the assembly of the manifold collar with the deep-well plate for the final elution step. Three reservoirs were used for storage of Wizard SV Lysis Buffer, wash solution and nuclease-free water.
Cross-contamination assessment
Cross-contamination is a potential issue when dealing with downstream applications involving PCR-based assays. Tissue cell culture and mouse tails samples were set up in checkered pattern to check for cross-contamination (Figure 3). Every other well in a 96-well tissue culture plate contained either cell culture media or cell culture media containing HeLa cells. For the mouse tail method, sample lysates were added to every other well (see Figure 3). The 96-well plate was subjected to the same genomic DNA purification protocol and PCR conditions described above with the resulting amplified products being visualized on agarose gels (see Figure 5).
Results and discussion
HeLa
The purity and integrity of the HeLa genomic DNA samples were examined by PCR amplification of the b-actin gene. The specific 285-bp PCR products were produced using HeLa genomic DNA samples (Figure 4). There was no detectable sample cross-contamination during sample preparation as shown with the sensitive PCR method using nine negative (wells with media only) samples (Figure 4) in the HeLa cross-contamination plate (Figure 3). The ability to specifically amplify a PCR product demonstrated that the Biomek FX and Wizard SV 96 Genomic DNA Purification could reliably isolate contaminant-free genomic DNA from adherent cells that would be suitable for many molecular biology applications.
Mouse tail lysate
The purity and integrity of the mouse tail genomic DNA samples were examined by PCR amplification of the IL1-b gene; amplification of the IL1-b gene generates a 1.2-kb band (Figure 5). There was no detectable sample cross-contamination during sample preparation as shown with the sensitive PCR method using five negative (wells with media only) samples taken from the mouse tail lysate cross-contamination plate (Figure 3). The ability to specifically amplify a PCR product demonstrated that the Biomek FX and Wizard SV 96 Genomic DNA Purification could reliably isolate contaminant-free genomic DNA from mouse tail lysate that would be suitable for many molecular biology applications.
Summary
Automation of the Wizard SV 96 Genomic DNA Purification System on the Biomek FX Liquid Handling System is fast and reliable. The ease in setting up the work surface contributes to the overall ease of use of the system. More importantly, as demonstrated here, this method reliably produces high-quality genomic DNA from different starting materials (cells and lysates), without detectable cross-contamination. The PCR analyses performed in this study demonstrate the purity of genomic DNA isolated with this system that may be used for a variety of downstream applications.
About the authors
Hobert Wai, Ph.D.; Matthew Cu; Dana P. Campbell; and Keith Roby are with Beckman Coulter, Inc. Terri Grunst and Daniel Kephart are with Promega Corp.
More information the products discussed in this article is available from the manufacturers. More information about the Biomek FX Liquid Handling System is available from: Beckman Coulter, Inc., Fullerton, CA. 800-742-2345;
www.beckmancoulter.com
The PCR process is covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann La Roche, Ltd. All trademarks are the property of their respective owners.
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