Method Note 008 PRODUCT CERTIFICATION Automation of the Macherey-Nagel NucleoFast 96 PCR Cleanup Kit on the Zephyr Genomics Workstation Introduction Here, we describe validation using Caliper’s Zephyr Genomics Workstation to automate PCR cleanup with the Macherey-Nagel NucleoFast 96 PCR Cleanup Kit (REF# 743500.4). Using this system, high quality PCR products are quickly purified with good yields by ultrafiltration on the Zephyr Genomics Workstation. DNA Yield and Quality Method 25 µL of a PCR reaction (PCR fragments sizes as indicated from 164 bp to 1484 bp) were diluted to 100 µL, loaded on NucleoFast 96 Plate. DNA was recovered in 100 µL nuclease free water. Unpurified or purified samples were dried in a Speedvac and dissolved in 15 µL loading buffer. Samples were analyzed on a 1 % agarose gel and by microfluidic capillary electrophoresis (CE). Figure 2. Agarose Gel Analysis of purified PCR products of 164, 252, 359 and 459 bp. (Tick marks indicate unpurified input samples) Figure 1. DNA Sequencing (982bp Fragment Shown): PCR fragments of 359bp, 459bp, 982bp and 645 bp were purified using the NucleoFast method. These PCR fragments are originally amplified from pBluescript DNA. For this we use a fixed forward primer (pBS_LN) and variable reverse primer to generate the products with different lengths in individual singleplex PCR reactions. Fragment ng/µL input/ no purification ng/µL purified % recovery 164 bp 252 bp 459 bp 782 bp 2.35 3.47 5.9 7.6 1.66 3.28 4.87 7.29 70 95 83 96 Table 1. Tabulated Microfluidic CE Yield Analysis of Selected Samples. Figure 3. Agarose Gel Analysis of purified PCR products of 645, 782, 982 and 1482 bp. (Tick marks indicate unpurified input samples) Figure 4. Microfluidic CE Analysis of Typical Unpurified 252bp PCR Product Sample Performance Volume Range: High Volume Head (HVH) 1 - 200 µL Precision: High Volume Head (HVH) 1 - 5 µL: CV <5% 5 - 200 µL: CV <2% 2 - 25 µL or 50 µL: CV 5% Weight: 75 kg (185 lbs) Base Unit Operating Temperature: 15 - 35 ºC (59 - 95 ºF) Operating Humidity: 0 - 85% RH, non condensing Figure 5. Microfluidic CE Analysis of Typical Purified 252bp PCR Product Sample Conclusion The Zephyr Genomics Workstation from Caliper Life Sciences can be used to automate PCR product purification with the Macherey-Nagel NucleoFast 96 PCR Cleanup Kit (REF# 743500.4). The process produces good yields of high purity DNA fragments, suitable for downstream applications (DNA sequence data not shown). The Zephyr Genomics Workstation intuitive, user friendly interface enables automated increased productivity with pre-programmed kits, requiring minimal training. Zephyr’s small foot print fits on to a standard lab bench, requiring no special table, while offering flexibility for molecular biology applications: • DNA extraction • RNA extraction • Plasmid isolation • PCR purification • Sequencing reaction clean-up • PCR setup • Sequencing reaction setup • DNA and RNA normalization Air Supply: Regulated 35 - 65 psi Power Input: 115 VAC, 50/60 Hz, 1000 VA max. or 230 VAC, 50/60 Hz, 1000 VA max. Caliper Life Sciences Part No. Description 125282 Zephyr Genomics Workstation with HV Head - 110V 125283 Zephyr Genomics Workstation with HV Head - 220V 119728 Optional Zephyr Environmental Enclosure 122000 LabChip GX Analyzer What’s included with your Zephyr GW • PC controller and monitor • Vacuum filtration station and pump • Waste bottle • Plate/lid gripper • Ultrasonic detector • Genomics Workstation GUI and Maestro software • Startup Kit ©2010 Caliper Life Sciences. All rights reserved. Caliper, the Caliper logo, Maestro, LabChip and Zephyr are tradenames and/or trademarks of Caliper Life Sciences, Inc. All other names are trademarks of their respective companies. www.caliperLS.com ZH-MN-008 Jul 10