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Focus: High-Throughput DNA Purification

Automated Isolation of Genomic DNA Using Promega’s Wizard® SV 96 DNA Binding Plates on the Beckman Biomek® 2000

This article demonstrates the use of Promega’s Wizard® SV 96 Binding Plate (Cat.# A2271) on the Beckman Biomek® 2000 for high-throughput genomic DNA isolation from multiple sample types.

By Terri Grunst, B.S.
Promega Corporation

Introduction

A high-throughput genomic DNA isolation procedure must provide for the isolation of high-quality DNA quickly. We have developed a robust procedure to isolate genomic DNA from many different sample types in a 96 well format using a Beckman Biomek® 2000. The procedure takes only one hour using Wizard® SV 96 DNA Binding Plates (Cat.# A2271) to purify genomic DNA from lysates of mouse tail clippings, plant tissue, tissue culture cells, whole blood or cheek swabs. A single automated procedure can be used regardless of the sample type. The genomic DNA isolated is of high quality and serves as an excellent template for PCR(a) analysis. Moreover, in these studies, no sample-to-sample contamination from the DNA purification procedure was detected.

Table 1. Protocols for Lysate Preparation from Indicated Sample Types.

Procedure

Mouse Tail

1. Digest each 0.5cm mouse-tail snip with 200µl Nuclei Lysis Solution, 48µl EDTA (0.5M) and 17.5µl Proteinase K (20mg/ml) solution. Vortex to mix.
2. Incubate for 3 hours to overnight at 55°C, mixing periodically.
3. After digestion, add 250µl SV RNA Lysis Buffer and vortex to mix.
Note: Lysates can be stored at -70°C

Plant Tissue
(Tomato Leaf)

1. Crush 100mg of plant tissue to a fine powder in liquid nitrogen with a mortar and pestle.
2. Add 1.5ml SV RNA Lysis Buffer to the tissue powder in the mortar after the liquid nitrogen has evaporated.
3. Crush the tissue further in SV RNA Lysis Buffer until tissue particles and fibers are no longer seen.
4. Transfer lysate to new 1.5ml microcentrifuge tube.
Note: Lysates can be stored at -70°C.

CHO Cells

1. Grow cells in a 96 well tissue culture plate at a density of 1 x 106 cells per well.
2. Wash cells once with 1X PBS.
3. Add 150µl SV RNA Lysis Buffer to each well. Mix by pipetting.
Note: Lysates can be stored at -70°C.

Whole Blood

1. Add 400µl of Lysis Buffer (SV RNA Lysis Buffer plus 1% Triton® X-100) to 200µl of whole blood. Vortex to mix.
Note: Lysate cannot be stored; use immediately after preparing.

Cheek Swab

1. Scrape the inside of the cheek with a clean pipette tip.
2. Swish the pipette tip in 150µl SV RNA Lysis Buffer plus 0.97% b-mercaptoethanol. Vortex to mix.
Note: Do not use a cotton swab to collect cheek sample. Cotton fibers from the swab will clog the Wizard® SV 96 DNA Binding Plate.

Automated Isolation of Genomic DNA

Sample lysate preparation will differ depending on the sample type for isolating genomic DNA. Table 1 briefly describes the lysate preparation for each sample type tested for this report. Once the lysate is prepared, genomic DNA isolation is performed on the Beckman Biomek® 2000, using a single protocol for all sample types.

For genomic DNA, 150µl of prepared lysate is placed into the well of a 96 well plate and the plate is placed on the deck of the Beckman Biomek® 2000. As shown in Figure 1, the remainder of the Biomek® 2000 deck is assembled according to the program’s initial deck configuration. Once configured, the Biomek® program is initiated and takes approximately one hour per 96 well plate (up to 96 samples) (see Table 2).

Table 2. Biomek® 2000 Program for Isolation of Genomic DNA.

Step

Process

1

Biomek® 2000 transfers 150µl of sample lysate from the 96 well sample plate to the Wizard® SV 96 DNA Binding Plate. The sample lysate is pulled through the plate by vacuum and the DNA binds.

2

The samples are washed twice with 500µl of Wizard® SV 96 Wash Solution containing 60% ethanol, using the Wash 8 tool.

3

The binding plate is dried briefly by vacuum.

4

The binding plate is transferred from the B6 to the A6 vacuum manifold position where it is placed on top of an elution plate fitted with a vacuum manifold collar.

5

DNA is recovered in the elution plate by addition of Nuclease-Free Water (75µl) to the binding plate.

Analysis

Purified genomic DNA is eluted in 75µl of Nuclease-Free Water (Cat.# P1193). For mouse tail, plant tissue and tissue culture cell samples, a 20µl aliquot was analyzed by agarose gel electrophoresis (Figure 2).

Quality of the genomic DNA was assessed by PCR. One microliter of the genomic DNA was used for PCR amplification in a 50µl reaction. Specific oligonucleotide primers were used to target genomic DNA sequences: IL-1b gene for mouse tail, b-actin for CHO cells, and a noncoding region of chloroplast DNA (1) for tomato leaf samples (Figure 3).

Sample cross-contamination was investigated by assaying an array of blood and cheek swab samples, alternating with water controls, in a 96 well plate. A prothrombin transcript was targeted by PCR in samples from all wells. As evident in Figure 4, no sample-to-sample contamination was observed in this experiment.

Discussion

An advantage of this automated genomic DNA isolation system is the flexibility of the procedure. In this study we isolated genomic DNA from a good representation of some common sample types. Types varied from those easily processed (e.g., tissue culture cells), to more difficult ones (e.g, whole blood and plant tissue).

Purified genomic DNA isolated using this automated procedure consists of largely intact genomic DNA of greater than 23kb. The isolated DNA contains trace amounts of RNA, which can be removed easily by adding 1µl of RNase ONE™ Ribonuclease (Cat.# M4261) directly to the eluted DNA or by adding 100µl per 10ml Nuclease-Free Water used to elute the DNA. The purified genomic DNA can be amplified by PCR and is suitable for use in many downstream applications. The processing time for isolation of genomic DNA is approximately one hour per 96 well plate, with no manual intervention. Multiple samples can be processed in one run with no cross-contamination.

A potential limitation of this automated genomic DNA isolation system is in processing viscous lysates. Sample lysates that contain excessive amounts of cellular debris will be viscous and will not move easily through the Wizard® SV 96 DNA Binding Plate. These samples may even clog the plate wells. For the samples processed in this study, the lysate preparation and solution volumes were optimized. Optimization should be considered for other samples to achieve successful genomic DNA isolation.

Conclusion

Genomic DNA can be isolated in a high-throughput format using Promega’s Wizard® SV 96 DNA Binding Plate on the Beckman Biomek® 2000. The automated procedure described here can be used to isolate quality genomic DNA >23kb that is ready for downstream applications. We demonstrated the flexibility of the procedure by isolating genomic DNA from multiple sample types. Importantly, no cross-contamination was detected using this automated procedure.

Reference

  1. Taberlet, P. et al. (1991) Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol. Biol. 17, 1105-1109.
(a)The PCR process is covered by patents issued and applicable in certain countries. Promega does not encourage or support the unauthorized or unlicensed use of the PCR process.
*Products may be covered by pending or issued patents. Please visit our patent and trademark web page for more information.
Wizard is a trademark of Promega Corporation and is registered with the U.S. Patent and Trademark Office. RNase ONE is a trademark of Promega Corporation.
Biomek is a registered trademark of Beckman Coulter, Inc. Triton is a registered trademark of Union Carbide Chemicals and Plastics, Co.

 

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Figure 1

 

Figure 1. Initial deck configuration of the Biomek® 2000.

 
Figure 2

Figure 2. Agarose gel electrophoresis of purified genomic DNA from mouse tail (MT), tomato leaf (TL) and CHO cells. 

 
Figure 3

Figure 3. Agarose gel electrophoresis of PCR products amplified from 1µl of mouse tail, CHO cells and tomato leaf samples. 

 
Figure 4

Figure 4. Cross-contamination analysis. 

 

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