Automation of the Macherey-Nagel NucleoFast 96 PCR Cleanup Kit on the Zephyr Molecular Biology Workstation (ZH-MN-008) (501 KB)

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
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