Abstract
Automating electrophoresis significantly reduces the time required for loading a large number of samples, increases the speed of electrophoresis analysis, and maximizes the resolution power (clear separation of fragments) of this technique. In addition, automation increases the precision of electrophoresis analysis. Here we demonstrate an automated, high-throughput method of loading 96 samples simultaneously onto an electrophoresis gel, using the Apogent Discoveries Tango™ system and the Invitrogen™ E-Gel® 96 system.
Introduction
Electrophoresis is a fundamental technique used to identify and separate DNA, RNA, and protein molecules. It is routinely used for quantification and determination of the quality of samples prior to performing downstream applications. The lack of a high-throughput automated method of performing gel electrophoresis analysis has created a major bottleneck in molecular biology applications such as sequencing, genotyping, SNP detection, and PCR analysis. Here we demonstrate an automated, high-throughput method of loading 96 samples simultaneously onto an electrophoresis gel using the Apogent Discoveries Tango systems 1 and the Invitrogen E-Gel 96 system.2,3 This procedure significantly reduces the time requirement for loading a large number of samples. Consequently, an increase in the overall speed of electrophoresis analysis is achieved. Furthermore, automation reduces sample-to-sample variability caused by errors in manually dispensing samples and as a result increases the precision of electrophoresis analysis.
Materials and Methods
A Tango system (Apogent Discoveries, Sunnyvale, CA) equipped with 96, 100μl standard syringes with DuraFlex™ needles was used for liquid handling. The E-Gel® 96 pre-cast 2% agarose gel (Cat. no. G7008-02), E-Gel® 96 holder (Cat. no. G7300-01), E-Gel® 96 mother base (Cat. no. G7100-01), BlueJuice™ gel loading buffer (Cat. no. 10816-015), 0.24-9.5 Kb RNA Ladder (Cat. no. 15620-016), and E-Gel® 96 Low Range DNA Marker (Cat. no. 12369-013) were provided by Invitrogen Corporation, Carlsbad, CA. PCR core kit (Cat. no. 1 578 553) and human genomic DNA (Cat. no. 1 691 112) were purchased from Roche, Mannheim, Germany. A human β-actin amplimer set (Cat. no. 5402-1) was purchased from Clonetech Laboratories, Palo Alto, CA. RNaseZap® (Cat. no. 970-82-84), was purchased from Ambion, Austin, TX. Samples were prepared in a Skirted CyclePlate-96 (Cat. no. 1047-20-0), Apogent Discoveries, Sunnyvale, CA and loaded following the procedures outlined in the E-Gel 96 high-throughput agarose electrophoresis system manual. ScreenMates™ (Cat. no. 4916) clear 96 well plates were from Apogent Discoveries, Sunnyvale, CA.
DISPENSING PRECISION OF THE TANGO LIQUID HANDLING SYSTEM
Prior to the use of the Tango system, the uniformity and consistency of the sample volumes dispensed across the arrays of syringes (96 syringes in this study) were determined by the coefficient of variance (C.V.) (http://www.apogentdiscoveries.com). In short, different volumes of a 10μg/ml fluorescein solution were dispensed into each well of a 96- or 384- well plate containing 0.1M Tris buffer. The final volume in each well after the fluorescein solution was dispensed was 100μl. Each plate was then read in a Tecan fluorescence plate reader and the C.V.s were determined across each plate for each of the dispensed volumes. A high uniformity for dispensing volumes equal to and higher than 100nl was evident with C.V.s of less than 4% (Table 1).
Dispensing precision of the Tango system.
OPERATING THE TANGO SYSTEM FOR LOADING DNA/RNA SAMPLES
The Tango system incorporates precision glass syringes (96 or 384) arrayed in standard SBS microplate spacing (Figure 1). Its stage is composed of 12 positions (nests). For this protocol, one nest was dedicated to the wash module, one to a reservoir containing 2% bleach, one to a reservoir containing deionized water, one to a Skirted CyclePlate-96 containing DNA or RNA samples (called the source plate), and one to an E-Gel 96 gel (placed on the E-Gel 96 holder). As many as four E-Gel 96 holders can fit onto the Tango's stage.
The Apogent Discoveries Tango system.
To clean the syringes and prevent carry-over contamination when dispensing DNA samples, a Tango protocol was created that incorporated three water wash cycles in the Tango wash module, one wash cycle with 2% bleach (from a bleach reservoir), followed by an additional three water wash cycles in the wash module (a total wash period of one minute and 20 seconds), before and after loading the E-Gel 96 gel (one “wash cycle” is defined as an aspiration and a dispense; in this instance, the wash volume was set at 20μl). Detailed procedures have been developed for cleaning syringes (decontaminating them from RNases, DNases, and proteins) before and after each dispense.4–6
For dispensing RNA samples, the Tango system was decontaminated by wiping the stage and the Tango wash module with RnaseZap, RNase-free water, and ethanol. The syringes were washed in the wash module for two wash cycles with a 2% bleach solution and then for three wash cycles with RNase-free water. Three wash cycles with 50% RNaseZap were performed next, followed by three wash cycles with RNase-free water. Finally, the syringes were washed with 100% ethanol for two cycles, and the syringes were emptied and left to dry overnight. This wash procedure takes a total of two minutes.
To load the DNA/RNA samples a Tango protocol was created to first preload the 96 well of the E-Gel 96 gel with 10μl of water (a 5μl of air gap followed by 10μl of water was aspirated into the syringes and then emptied into the wells of the gel). According to the manufacturer's (Invitrogen) instructions, although preloading of the gel with 10μl of water increases the precision of electrophoresis, the preloading step can be omitted. Next, the samples (from the Skirted CyclePlate-96) were loaded onto the E-Gel 96 gel (a 5μl air gap followed by 10μl of sample was aspirated into the syringes and then emptied into the wells of the gel). The loading of the electrophoresis gel takes a total of 15 seconds using the Tango system (versus five minutes if loaded manually). Once the samples were loaded, the E-Gel 96 gel was transferred to the E-Gel 96 mother base to begin electrophoresis (the voltage parameters are preset into the system and therefore no manual manipulation is required). When electrophoresis was complete (electrophoresis time of approximately 12 minutes), the gel results were visualized and photographed under ultraviolet light.
Results and Discussion
Using the Tango system, DNA and RNA samples were loaded onto E-Gel 96 gels. The loading time, including the time required for the process of priming (trial dispensing required for a higher dispensing precision), was approximately 15 seconds. The electrophoresis time for the DNA (0.1-2.0 kb) and RNA ladder (0.24-9.49 kb) was approximately 12 minutes. Figure 2a shows that DNA samples loaded traveled as a single high-resolution, high-quality band. Figures 2b shows the exceptional quality of separation between different lengths of DNA fragments. No electrophoresis flaws, such as diffusion of the sample, smearing, or tailing were detected.
Electrophoresis result using the Tango automated liquid handling system and the Invitrogen E-Gel 96 system. Sample: 10μl (50ng) of β-actin PCR product (length: 838bp), DNA marker (50ng, length: 100, 200, 400, 800, 2000 bp): E-Gel 96 low range DNA marker.
Electrophoresis result using the Tango automated liquid handling system and the Invitrogen E-Gel 96 system. Sample: E-Gel 96 low range DNA marker (90ng, length: 100, 200, 400, 800, 2000 bp).
In addition to the loading of DNA samples, polyA-tailed RNA samples were also successfully loaded onto the E-Gel 96 gel using the Tango system. As indicated in Figure 2c, no RNA degradation was observed when samples were loaded with the Tango system. These results demonstrate a simple, fast, and precise method for automating electrophoresis.
Electrophoresis result using the Tango automated liquid handling system and the Invitrogen E-Gel 96 system. Sample: 150ng RNA ladder (240, 1350, 2370, 4400, 7460, 9490 bp).
Conclusion
The Apogent Discoveries Tango system and the Invitrogen E-Gel 96 system work together to provide a fast, simple, precise, and automated method for simultaneously loading and analyzing a large number of samples for high-throughput electrophoresis.