improving the molecular biology workflow
TRANSCRIPT
Streamlining the Molecular Biology Workflow
Labnet International Presents
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The Molecular Biology Workflow
The Molecular Biology Workflow Involves 4 Key Steps:
1. Sample Prep2. PCR Prep3. PCR4. Validation
Overview
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Liquid Handling is needed throughout the Molecular Biology workflow, from Sample Prep to PCR.
Reliable and accurate liquid handling is critical.
Liquid Handling
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• Personnel → Good pipetting technique comes from practice
• Facility → Environmental conditions• Apparatus → Right instrument• Procedures → Regular Maintenance • Material and reagents → Performance
checks (calibration)
Good Pipetting Procedures
Liquid Handling
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SET THE VOLUME
• Using a push button or a knob
• Always possible with one or two hands and with gloves
AVOID PARALLAX ERROR
• Horizontal position• Volume lock• Approach a volume from
a higher value
Setting the Volume
Liquid Handling
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ASPIRATE AND DISPENSE BACK BEFORE USE!
Why?• Greater uniformity• Great accuracy and
precision
When?• After changing the tip• After increasing the
volume
Pre-rinsing – Wet Tip
Liquid Handling
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1. Results obtained by untrained technician (red curve)
2. Results obtained by a trained technician (green curve)
• T: Target – Set Volume• R: Volume range –
permitted by the manufacturer specification
Personnel - User Mistakes Influence Results
Liquid Handling
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1. Sample Prep
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Prism™ R Refrigerated Microcentrifuge
• Powerful Refrigeration attains 4°C in just 8 minutes
• Compact footprint - only 25.4 cm wide
• Aluminum rotor• 24 x 1.5/2.0 ml tubes
or PCR strips• Easy Access Design• Optional Strip Spin
Adapter
Sample Prep
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Axygen Mini-Prep Multisource Genomic DNA Kit
• Mammalian Tissue• Plant• Cells• Fungi
Sample Prep
Transfer Miniprep column to a 2 ml microfuge tube and centrifuge for 1 minute at 12,000 x g.
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Prism™ R Easy Tube Access
Samples are easy to insert and remove from the rotor.
Sample Prep
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Prism™ R Rapid Acceleration and Deceleration
Maximum G-Force: 17,135 x gMaximum RPM: 13,500 rpm
Sample Prep
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Prism™ R Reduces Rotor Handling
24 x 1.5/2.0ml aluminum rotor
Sample Prep
24 x 1.5/2.0ml aluminum rotor
Additional adapters are available for spinning tubes smaller than 1.5ml
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2. PCR Prep
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IMPROVES REPRODUCIBILITY AND STREAMLINES WORKFLOW FEATURES AND BENEFITS
• Quickly spins down droplets and condensation
• Max RPM = 2,500• Max G = 500 x g• Use before and after PCR
to improve yield and reduce noise
• Accepts skirted, non-skirted and all standard PCR plates
• Less than ¼ the size of standard plate centrifuges
MPS 1000 Mini Plate Spinner
PCR Prep
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LESS THAN ¼ THE SIZE OF STANDARD PLATE CENTRIFUGES
MPS 1000 Mini Plate Spinner – Size Advantage
PCR Prep
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Application: MPS 1000 Mini Plate Spinner Centrifuge for Real Time PCR
Studies show that using the MPS 1000 before PCR increases PCR yield and reduces “noise” in the dilution series.
PCR Prep
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MPS 1000 Mini Plate Spinner – How it Works
1. Insert the Plates2. Spin for 20 seconds3. Remove the Plates
PCR Prep
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Plate Adapter for MPS 1000 Mini Plate Spinner
Supports:• 8 x 0.2ml PCR Strips• 12 x 0.2ml PCR Strips• 0.2ml PCR Tubes• 4 x 6 half plates
PCR Prep
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3. PCR
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AccuSeal™ Semi-Automated Plate Sealer
The new Labnet AccuSeal is a semi-automated heat sealer designed to seal a wide variety of microplates using different types of heat seal films.
PCR
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• Consistent sealing across all wells• Consistent results plate to plate• Less chance of leakage or evaporation• Better integrity at low temperatures (-80°C
freezer storage)• A variety of types of seals for flexibility with
applications• Easy to operate – set the parameters
correctly and consistent results are virtually guaranteed
Advantages of Thermal Sealing
PCR
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• PCR Thermal Cycler• 96 well format• Fast run times• “Better than
Gradient”• Upgrade of
MultiGene Gradient• 3 year warranty
MultiGene™ OptiMax Thermal Cycler
PCR
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MultiGene™ OptiMax – Custom Optimization – “Better than Gradient”
PCR
1. Can set all wells for 1 temp2. Gradient above sets 1st
column and last column and cycler determines values in between
1. Can set all wells for 1 temp2. Gradient above sets 1st block and
last block and cycler determines values in between
3. User can set values of choice for each block – “Better than Gradient”
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AS PUBLISHED IN MARCH 2013 BIOTECHNIQUES
Streamlining the Genotyping Workflow with the MultiGene™ OptiMax
PCR
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P GENE
• 94°C for 3 minutes
35 cycles of:• 94°C for 30
seconds• 51.7°C for 1
minute• 68°C for 1 minuteThen• 68°C for 2
minutes• 4°C hold
MultiGene™ OptiMax
PCR
D GENE
• 94°C for 3 minutes
35 cycles of:• 94°C for 30
seconds• 61.0°C for 1
minute• 68°C for 1 minuteThen• 68°C for 2
minutes• 4°C hold
T GENE
• 94°C for 3 minutes
35 cycles of:• 94°C for 30
seconds• 65.0°C for 1
minute• 68°C for 1 minuteThen• 68°C for 2
minutes• 4°C hold
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Comparisons of results with Traditional Thermal Cycler and OptiMax
PCR
1- Ladder2- WT (wild type)3- P transgene + WT4- P transgene + WTControls were from previously genotyped animals displaying either a P transgene or a Wild Type (WT) band. (Not depicted here)
1- Ladder1 – F1/F1 (homozygous of T gene)3- Het (one copy of T gene + WT)4- F1/F1 (homozygous of T gene)Controls were from previously genotyped animals displaying either a WT or both WT gene bands. (Not depicted here)
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Comparisons of results with Traditional Cycler and OptiMax
PCR
1- Ladder2- DR/DR (homozygous of D gene)3- Het (one copy of D gene + WT)4- HETControls were from previously genotyped animals displaying either a homozygous for D gene or both WT and D gene bands. (Not depicted here)
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SIMPLIFIED WORKFLOW – IMPROVED THROUGHPUT
The MultiGene OptiMax thermal cycler may be programmed to operate between one and six different annealing temperatures of user choice across the block.• Improved throughput by
reducing time• Simplified workflow by
reducing steps
MultiGene™ OptiMax
PCR
Fig. 1 Primers designed to anneal to various genes (e.g Gene A, B, and C) typically have different annealing temperatures (T1,T2 and T3). To simplify workflow and increase throughput, the MultiGene OptiMax can perform up to six different reactions at the same time thus reducing the time from 9+ hours to three.
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Better than Gradient cyclers are ideal for Genotyping labs:1. Zebra fish2. Arabidopsis3. Drosophila4. Transgenic Mice
Technical / Applications
PCR
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4. Validation
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IMPROVES SPEED AND REDUCES SETUP TIME
1. Saves time2. Run entire 96 well plate
for quick screening3. Multichannel pipette
compatible
Enduro™ 96 Horizontal Gel Box
Validation
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SPEED AND EASE OF USE
1. Blue light LEDs – Powerful EPI blue LEDs enable the user to capture beautiful images of SYBR safe stained gels
2. Tablet interface creating small footprint – system arrives fully assembled, with software preinstalled.
3. 3 position filter slider – increased application flexibilty – orange filter comes standard (green, red and blue filters are also available.)
Enduro™ GDS Touch Gel Documentation System
Validation
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SPEED AND EASE OF USE
1. 4 clicks to results2. Small footprint3. Improved blue light
sensitivity4. Will never need to touch
camera5. Improves workflow
Enduro™ GDS Touch Gel Documentation System
Validation
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GEL IMAGED WITH BLUE LIGHT CONVERSION SCREEN
GEL IMAGED BLUE LED – ENDURO™ GDS TOUCH
Enduro™ GDS Touch Applications
Validation
Blue LED provides greater sensitivity as a Blue Light Conversion Screen SYBR® Safe
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• DNA gels• Protein gels• Stains including but not limited to:
• Ethidium Bromide• Coomassie Blue• Silver Stain• SYBR® Gold• SYBR® Green• SYBR® Safe• GelStar• SYPRO® Ruby
Enduro™ GDS Touch Applications
Validation
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1. Centrifugation – Reduces cool down time
2. Liquid Handling – Good practices and easy self calibration
3. Plate Spinner – More reproducible results
4. Plate Sealer – More reproducible results
5. Thermal Cycler – Reduced time and steps
6. Gel Electrophoresis – Reduced preparation time
7. Gel Imaging – Reduced run time and improved results
Streamlining Molecular Biology Workflow
Conclusion
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Learn More About Streamlining the Molecular Biology Workflow. Visit us at http://labnetinternational.comTwitter: @labnetnews