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Bob McLaren, Ph. D., Promega CorporationPromega has recently developed a validated method for isolating DNA samples on the Biomek 3000 using the DNA IQ system. The Biomek 3000 provides additional functionality not found in the older Biomek 2000, such as a more flexible software package that allows a single method to process multiple sample types and sample numbers. Sample tracking and on-the-fly well picking are also available. Unlike the previous Biomek 2000 method, which used the Thermal Exchange Unit heated with recirculating water and the MicroMix 5 shaker, the Biomek 3000 method uses a new electrical heater and a separate shaker. In addition, the Greiner U-bottom collection plate, which was previously used with the Biomek 2000 method, has been replaced with a 1.2 mL round-well deep-well plate. This allows more vigorous shaking during the wash and elution steps with the new shaker without risk of solutions splashing out of the wells and contaminating adjacent wells. To facilitate heating of this deep-well plate during the elution step, Promega also developed a new deep-well heat transfer block.
New Hardware and Plasticware to Improve EfficiencyPromega has integrated four new pieces of hardware on the Biomek 3000. These items are the V&P Scientific Heating Block (Promega Cat.# V6761), the Deep Well Heat Transfer Block (Promega Cat.# 6741), the Variomag Teleshake Shaker (Promega Cat.# V6751) and Shaker Integration Plate (Promega Cat.# V3691) for securing the shaker on the deck of the Biomek 3000 (Figure 1). Both the Variomag Teleshake shaker and the V&P Scientific Heating Block sit on the right hand expansion module of the Biomek 3000. There is no need for a separate integration unit in place of the right hand expansion module as was the case for the MicroMix 5 shaker. A 1.2 mL Round-Bottom Deep Well Plate (Promega Cat.# V6771) is now used for processing the samples. This replaces the Greiner U-bottom collection plate that was previously used with the Biomek 2000 method. Figure 2 shows the deck layout of the Biomek 3000 for processing 96 samples using the Promega DNA IQ System.
The new Deep Well Heat Transfer Block is placed on top of the V&P Scientific Heating Block at position A6 on the deck of the Biomek 3000. The Variomag Teleshake Shaker is placed at position B6 and held in place by the Shaker Integration Plate (not visible in this picture).
In addition to the V&P Scientific Heating Block and the Variomag Teleshake Shaker, the deck setup on the Biomek 3000 for processing 96 samples with the DNA IQ System requires four boxes of P250 barrier tips (positions A2 to A5), a reservoir containing reagents at position B3, a 2 mL square-well deep-well plate containing samples at position B4, and a 1.2 mL Round-Bottom Deep Well plate on a MagnaBot® 96 with a ¼ inch foam spacer at position B5. An elution plate is placed at position B1. The tools used are the MP200 8-channel pipetting tool and the gripper tool. Heating Profile With New Heating Block and Deep Well Heat Transfer BlockDNA is eluted from the DNA IQ Resin by adding DNA IQ Elution Buffer to the resin and heating at 65°C for 5 minutes. The heating is required for efficient elution. To determine the efficiency of the heat transfer with the new hardware during the elution step, we measured the temperature of 100 µL of DNA IQ Elution Buffer (the standard elution volume) in three separate wells of a 1.2 mL Round-Bottom Deep Well Plate during the elution step of the method. The elution step consists of two 2.5-minute incubations of the 1.2 mL Round-Bottom Deep Well Plate on the Deep Well Heat Transfer Block with the V&P Scientific Heating Block set at 85°C with two 30-second shaking steps after each heating step. The data are shown in Table 1. The results clearly show that the temperature in the wells easily reaches 65°C by the end of the second 2.5-minute incubation with little drop in temperature during the final 30-second shake step.
Table 1. Temperature Profiles in Wells of the Deep Well Heat Transfer Block. The temperature of 100 µL of DNA IQ Elution Buffer was determined in three separate wells of a 1.2 mL Round-Bottom Deep Well Plate at each step of the standard elution procedure for the Biomek 3000 method. The V&P Scientific Heating Block was set at 85°C. Baseline denotes temperature in the well before placing the plate on the heater. Method PerformanceTo evaluate cross contamination with the Biomek 3000 method, cotton buccal swabs were processed in a checkerboard pattern with blanks consisting of DNA IQ Lysis Buffer. Samples were eluted in DNA IQ Elution Buffer, and the swab and blank samples were used in a standard 25 µL amplification using the PowerPlex 16 System. Representative results from one electropherogram each for a swab and blank sample are shown in Figure 3. No detectable cross contaminating peaks were seen in the blank samples even when a tenfold higher volume of eluted blank sample compared to swab sample was amplified.
Cotton buccal swabs and blanks were preprocessed in a checkerboard pattern on the Biomek 3000. Samples were amplified using PowerPlex 16 and analyzed on an ABI PRISM 3130 Genetic Analyzer. Ten times as much blank sample was amplified as swab sample. The top panel shows a representative profile from a buccal swab. The bottom panel shows a representative profile from a blank well. Conclusions
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Additional Information
For comments or questions about T3 Update, please contact David Daniels, Ph.D., editor.
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.jpg "New integration hardware for the Biomek 3000 method using the DNA IQ System")
.jpg "Deck layout for the Biomek 3000 method using the DNA IQ System")
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