Just another DNA extraction system? Liquid handling marries automated Centrifugation Dr. Jürgen Zimmermann Genomic Core Facilities EMBL, Heidelberg 2004/03
Dec 23, 2015
Just another DNA extraction system?
Liquid handling marries automated Centrifugation
Dr. Jürgen ZimmermannGenomic Core Facilities EMBL, Heidelberg
2004/03
DNA Extraction from primary sources may be complex
Laboratory NeedsRobust process, which can cope with a broad range of even viscous samples conditions
Consistency in DNA yield & DNA quality
DNA should be used PCR, restrictions, cloning
Flexible hardware should allow downstream applications or foreign processes
Fully automated process
Expandable
Scalability
Laboratory Specs
DNA yield: up to 20µg–40µg(animal/plant)
DNA size: >30kBp
DNA Purity: 1.8-2.0 (OD260/280)PCR, Digests
Reproducibility: ~5% CVSpeed: ~192samples/2h (Offline Lysis)
Chemical Concepts
Beads (Magnetic / Silica)Anion Exchange ChromatographyOrganic Extractions & PrecipitationsContaminant Binding in Filter materialsSilica Filters
NucleoSpin 96 Tissue HC MACHEREY-NAGEL, DürenNucleoSpin 96 Plant HC MACHEREY-NAGEL, Düren
Automation Concepts
Vacuum Chambers
Magnetic Bead Separators
Semi-automated Approaches
Dedicated instruments
Liquid handling System & Automated Centrifuge
Development based on MPII, PerkinElmer
Current Status: Automated DNA extraction from complex samples
Vacuum filtration with limited robustness(clogging, cross-contamination)
Magnetic beads with limited performance(time consuming, liquid handling in viscous solution, carryover)
Contaminant binding with limited specificity & capacity(time consuming, centrifugation speed limited)
Dedicated instruments with limited applicability & flexibility
The Instrument
Online Incubation
Shaking
Centrifugation
Gripper
FeaturesComplete Integration(Including sample digestion)Independent operation of centrifuge & liquid handling systemModular ConceptSample throughput 192/hRCF up to 6.300gOpen to any chemistryBasket Concept
Software
Process Window Deck Layout
Output Input
Incubators
Buffers
Applications
DNA extraction from animal tissues(mouse tails, mouse embryos, fish embryos, worms) [available]DNA extraction from plant tissues(various plants and organs) [available]Precipitation & Solubilisation ExperimentsSPEBAC DNA purificationProtein purificationChemical Synthesis
Process for Animal Tissues I
Process for Animal Tissues II
Mouse Tails: DNA Yield & Size
DNA extracts from 4 mm mouse tail tips using NucleoSpin 96 Tissue HC, 0.8% agarose gel. 1/15 of the eluate is loaded. /Hind III marker
Mouse Tails: Quality & Reproducibility
Genomic DNA from mouse tail clippings is prepared using NucleoSpin® 96 Tissue HC on Spinner. 2µl of each 1:10 dilution of 16 DNA eluates are used as template in a 20µl amplification reaction (40 cycles) in a LightCycler™ analysis (SYBR® Green). The PCR product generated is 212 bp for the Mus musculus cytoplasmic aconitase exon (acoI).A single amplification product is obtained (see melting curve). The obtained Crossing points for all 16 samples show a mean value of 26.02+/- 0.29 (CV 1.12%).
Cross Contamination Test
Mouse tail clipping samples and water are arrayed in adjacent wells of a 96-well plate. Genomic DNA is isolated using NucleoSpin 96 Tissue HC on Spinner. 2µl of each eluate is used for PCR (35 cycles) of Mus musculus cytoplasmic aconitase exon (acoI) (212 Bp). PCR product from 10µl of each PCR assay are separated on a 1 % agarose gel. No PCR product is detected in water samples demonstrating the absence of cross-contamination.
Plant Tissues: DNA Size & YieldWheat leafMaize leaf
Soybean leaf Sunflower leaf
DNA extracts from plants using NucleoSpin 96 Plant HCSample: 50 mg plant leaf - Elution volume: 150 µl CE- 1 % TAE agarose gel10µl (wheat, maize) / 15µl (soybean, sunflower) /Hind III Marker
Plant Tissues: Digestibility
DNA isolated from different plant species (digested, undigested)Sample amount: 50mg. Elution volume: 150µl CE. 10µl (wheat / maize) or 15µl (soybean / sunflower) of the eluate were run after digestion with EcoRI (A) or undigested (B) on a 1 % TAE agarose gel. M1: /HindIII, M2:1 kb ladder
1 2
wheat, leaf
M A B A B
maize, leaf sunflower, leaf
M A B A B
soybean, leaf
1 2
Plant Tissues: PCRmaize seed, leaf wheat seed, leaf
sunflower seed, leaf soybean seed, leaf
Amplification of a 750bp fragment from the nad5 gene
Achieved Technical Objectives
Robust application for DNA extraction from various complex sources were developedDNA quality compatible for RT-PCR, restriction digests, southern & cloningFully Automated system for automated centrifugation, liquid handling, incubation and shakingLiquid handling system and centrifuge can be used independentlyIntegrated multi-utility instrument
Achieved Lab Objectives
Reduced hands on time (load deck)
No human supervision necessary
Increased reproducibility
Modularity
Multipurpose Solution
Acknowledgements
MACHEREY-NAGEL Thomas Zinn Markus Meusel Ulrich Schübel
Inheco Bernhard Loibl
Hettich Klaus Günter-Eberle
EMBLEM Martin Raditsch Gabor Lamm
PerkinElmer Ralf Griebel Marek Turewiz Paul Lomax Martin Long
EMBL David Ibberson Leo Burger Wolfgang Dilling Vladimir Benes Christian Boulin