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[h=2]Introduction[/thanks]DNA of high purity and integrity is needed for many molecular-based applications including microarrays, DNA sequencing, PCR and Southern blotting. The increasingly higher throughput rates required for these applications has led to the development of novel technologies and chemistries for isolating DNA.
Solid phase reversible immobilization (SPRI) technology using magnetic bead-based kits has been shown to be a cost effective way of isolating DNA and can be used with automated liquid handlers. This technique is based on the interaction between the sample DNA and the ligand (silica or carboxylic acid) that coats the surface of the magnetic bead.
have validated their VERSA 1100 Nucleic Acid Isolation and PCR setup Workstation with AB96 Magnetic Bind Blood DNA Isolation kit to provide an ideal solution for this application.
[h=2]Objectives of the Experiment[/thanks]The goal of the experiment was to achieve the hands-free processing of blood samples, excellent gDNA yields, and the isolation of high quality gDNA that was intact, bankable, and of high molecular weight and high purity. The isolated gDNA also needed to be ready to use in a PCR setup and compatible with other downstream applications.
[h=2]Methods and Materials[/thanks]The isolation kit used was the AB96 Magnetic Bind Blood DNA Isolation kit (Figure 1) from . For sample preparation, the original samples of pig blood were placed in 48 wells on the deck of the workstation that was equipped with a 96-well-microplate (n=48), reagent reservoir, magnetic block, plate cooler, 96 channel aspirator and loaded with tip boxes (Figure 2).
[h=2]Results and Discussion[/thanks]The data indicated that liquid handling during the isolation process was efficient and consistent in the VERSA Workstation, maximizing DNA recovery as well as the uniformity of the DNA quality from the samples. High molecular weight gDNA migrated close to 23kb standard molecular hyper DNA ladder and no gDNA smearing was detected in the lanes of the agarose gel on ethidium bromide staining, as shown in Figure 4.
Furthermore, AbS26o/28o values were between 1.80-1.91, suggesting high purity of the isolated DNA, with no significant amount of protein and RNA detected. Low variation in CV% among these values also indicated that all the blood samples were handled in the same way throughout the automated process. Moreover, the Abs260 readings showed no gDNA in either the wash or the second elution.
The gDNA was highly representative because an amplicon from the house-keeping gene β-actin was amplified from 16 gDNA extracts that were randomly selected. These amplicons all gave clear, intense bands. The absence of smears and additional bands suggests that false positive amplification or mispriming does not occur. This successful amplification also indicates that the use of Aurora's Alcohol Removing Wash Buffer (ARW) provides an effective substitute to alcohol drying in the post-alcohol wash step.
[h=2]Conclusion[/thanks]Aurora's VERSA 1100 Workstation provides a cost-effective solution for reproducible and efficient isolation of high quality gDNA that is characterized by high yield, high purity and high molecular weight. This workstation is suitable for PCR applications and any other applications requiring high quality gDNA.
[h=2]Acknowledgement[/thanks]Produced from articles authored by Sikander Gill PhD, Rajwant Gill PhD, Sunyongna MSc, and Dong Liang PhD.
[h=2]References[/thanks]
Aurora's product range includes automated liquid-handling equipment, atomic absorption spectrometers, atomic fluorescence spectrometers, ion channel screening technology and microwave digestion systems, increasing the efficiency of sample management in a wide range of research applications. We are headquartered in Vancouver, BC, Canada, and have global sales, support, and service offices.
Solid phase reversible immobilization (SPRI) technology using magnetic bead-based kits has been shown to be a cost effective way of isolating DNA and can be used with automated liquid handlers. This technique is based on the interaction between the sample DNA and the ligand (silica or carboxylic acid) that coats the surface of the magnetic bead.
have validated their VERSA 1100 Nucleic Acid Isolation and PCR setup Workstation with AB96 Magnetic Bind Blood DNA Isolation kit to provide an ideal solution for this application.
[h=2]Objectives of the Experiment[/thanks]The goal of the experiment was to achieve the hands-free processing of blood samples, excellent gDNA yields, and the isolation of high quality gDNA that was intact, bankable, and of high molecular weight and high purity. The isolated gDNA also needed to be ready to use in a PCR setup and compatible with other downstream applications.
[h=2]Methods and Materials[/thanks]The isolation kit used was the AB96 Magnetic Bind Blood DNA Isolation kit (Figure 1) from . For sample preparation, the original samples of pig blood were placed in 48 wells on the deck of the workstation that was equipped with a 96-well-microplate (n=48), reagent reservoir, magnetic block, plate cooler, 96 channel aspirator and loaded with tip boxes (Figure 2).
Figure 1. AB96 Magnetic Bind Blood DNA Isolation kit Cat # MB-7899.
Figure 2. Deck of VERSA 1100 NAP/PCR Setup Workstation.
The automation protocol was performed as shown in Figure 3. The 96 channel aspirator was used to shorten the duration of the isolation process. The VERSAware software was used to define the automation workflow.
Figure 3. Automation protocol and workflow for gDNA isolation and PCR setup using Versa 1100 NAP/PCR Setup Workstation.
PCR setup was carried out using the isolated gDNA for amplification of β-actin and thermocycling was performed off the deck using a MiCycler Thermal Cycler. Finally, the quality of the isolated gDNA (its purity and recovery) was assessed using agarose gel electrophoresis and Abs26o/28o using a Bio-Tek HT Multi-Mode Microplate Reader.[h=2]Results and Discussion[/thanks]The data indicated that liquid handling during the isolation process was efficient and consistent in the VERSA Workstation, maximizing DNA recovery as well as the uniformity of the DNA quality from the samples. High molecular weight gDNA migrated close to 23kb standard molecular hyper DNA ladder and no gDNA smearing was detected in the lanes of the agarose gel on ethidium bromide staining, as shown in Figure 4.
Figure 4. Gel electrophoresis of 32 randomly selected gDNA extracts obtained with the VERSA 1100 NAP/PCR Setup Workstation.
In addition, the brightness of the DNA bands was of equal intensity, indicating reproducibility and consistency in liquid handling. The Abs260 readings further supported this by providing consistent sample concentrations among different samples in the range of 10.0-15.0ng/pL, which is satisfactory for most downstream applications.Furthermore, AbS26o/28o values were between 1.80-1.91, suggesting high purity of the isolated DNA, with no significant amount of protein and RNA detected. Low variation in CV% among these values also indicated that all the blood samples were handled in the same way throughout the automated process. Moreover, the Abs260 readings showed no gDNA in either the wash or the second elution.
The gDNA was highly representative because an amplicon from the house-keeping gene β-actin was amplified from 16 gDNA extracts that were randomly selected. These amplicons all gave clear, intense bands. The absence of smears and additional bands suggests that false positive amplification or mispriming does not occur. This successful amplification also indicates that the use of Aurora's Alcohol Removing Wash Buffer (ARW) provides an effective substitute to alcohol drying in the post-alcohol wash step.
[h=2]Conclusion[/thanks]Aurora's VERSA 1100 Workstation provides a cost-effective solution for reproducible and efficient isolation of high quality gDNA that is characterized by high yield, high purity and high molecular weight. This workstation is suitable for PCR applications and any other applications requiring high quality gDNA.
[h=2]Acknowledgement[/thanks]Produced from articles authored by Sikander Gill PhD, Rajwant Gill PhD, Sunyongna MSc, and Dong Liang PhD.
[h=2]References[/thanks]
- Hukari et al. J Lab Autom. 2011;16(5):355-365.
- Hellberg et al. J Lab Autom. 2011;16(4):308-321
- Broutian et al. J. Clin Virol. 2011;50(4):270-275.
Aurora's product range includes automated liquid-handling equipment, atomic absorption spectrometers, atomic fluorescence spectrometers, ion channel screening technology and microwave digestion systems, increasing the efficiency of sample management in a wide range of research applications. We are headquartered in Vancouver, BC, Canada, and have global sales, support, and service offices.
