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Overview of Bindley Bioscience Center Protein Production Lab: experimental capabilities. Bindley Biosceince Center, room 222 Phone 496-3102 http://www . ...? is coming Larisa Avramova. Contact. PCR plate. Filtration plate. Deepwell plate. Multiple samples? Repeating assays? - PowerPoint PPT Presentation
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Overview of Bindley Overview of Bindley Bioscience Center Bioscience Center
Protein Production Lab:Protein Production Lab:experimental experimental capabilities.capabilities.
Contact
Bindley Biosceince Center, room 222Phone 496-3102http://www. ...? is comingLarisa Avramova
Multiple samples? Repeating assays?
Tired of manual pipetting?
We can help!
Running of experiment with many samples in parallel (high throughput, HTP) requires the special labware: the 96 well plates. Different types of plates exist – PCR, filtration, deepwell.
That is why all instruments in the our lab have to be outfitted to handle plates. And they are! PCR plate
Filtration plate
Deepwell plate
Standard
Equipment designed for High Throughput experiments:1. Beckman HighThroughput Liquid Handling System “
BiomekFX”.Dual arm for 8-tip and 96–tip pipettors,Filtration module,Absorbance-plate reading module,8 Modules for fixing 96-wells plate to the instrument2 cooling-heating modules,Waste disposal module.
2. MJResearch PCR Thermocycler “DNA Engine DYAD” (gradient available).
3. Sigma-Quiagen Centrifuge 4K15C with rotor for 96-well plates.
4. Amersham Biosciences Liquid Chromatography system.
AKTAexplorer100 ,
Autosampler A-905,
Frac-950 fraction collector with rack for 96-well plate,
Midwest Scientific Chromatography refrigerator.
5. Kuehner AG Cryo replicator System “Duetz”.
6. Ready-to-run electrophoresis systems for 96 sample of DNA, Invitrogen,Vertical and ready-to-run PAGE-SDSsystems for 26, 52 and 96 sample of protein, Invitrogen, BioRad
min 0.2ul max 1200ul
“Matrix”
Max 1250ul min 1ul
7. Multichannel pipettors.
“Biohit”
8. Thermomixer, shaker, heated/refrigerated incubator with holders for microtiter plates.
Rocking platform
Vortex
Orbital shaker
Thermomixer
Benchtop Model Shaker-Incubator“Innova 4230”
Applications Applications (including but not limited to)(including but not limited to)
High-throughput cloning: Generation of PCR fragments. Analysis by agarose gel. Digestion with cloning enzymes. Ligation of fragments. Bacterial transformation and growth (plasmid propagation). Isolation of plasmid DNA. Verification of correct plasmid.
Screening for soluble protein: Transformation (protein expression). Screening of growth conditions. Preparation of soluble/insoluble fraction and their analysis by PAGE.
Custom design of assays and experiments to suit your particular high-throughput needs
Give us a
call t
o discuss
your optio
ns.
Design of ConstructsDesign of Constructs•Different types of tag: His, MBP, GST, Trx, •Different location of tag: N- or C-termini•Different size: full length or truncated protein •Different sources
Ligation Bacterial transformation and growth
(plasmid propagation) Plasmid DNA isolation, verification of correct
plasmid Bacterial transformation and growth
(protein expression) Screening for soluble protein
Purification of protein
Standard Steps for Cloning Standard Steps for Cloning ExperimentExperiment
Insert preparation Primer design PCR fragment generation Digestion with cloning
enzymes
Vector preparation Isolation of plasmid
vector Digestion with cloning
enzymes
Target choosing
Bacterial transformationBacterial transformation Chemical Heat-shock
PCR AmplificationPCR Amplification
~1500bp~500bp
Invitrogen, Precast 1% agarose gel
Q-tray
Procedure for plasmid purificationProcedure for plasmid purification96 x 1–1.25 ml E. coli bacterial culture in 96-Deepwell Culture PlateCentrifuge Deepwell Culture Plate. Pour off or aspirate supernatants Add Resuspension buffer.
Add Lysis buffer. Add Neutralization buffer
Pipette each lysate in the Culture Plate to Filter Plate A. Apply vacuum.
Add DNA Binding Buffer to Filter Plate DB (on top of Manifold). Apply vacuum .Add Washing buffer to Filter Plate DB. Apply vacuum.
Add Molecular Biology Grade Water to DNA binding matrix in Filter Plate DB. Incubate.Gradually apply vacuum to elute plasmid DNA into Collection Plate Result: purified plasmid DNA in Collection Plate
Place Filter Plate A on top of FilterPlate DB
Place Filter Plate DBon top of Collection Plate
Protein expressionProtein expression
LB media
autoinduction IPTG induction
18 C 37C Low IPTG High IPTG
18C
25C
18C
25C
37C 37C
25C
Screening for Soluble ProteinScreening for Soluble Protein
Separate phases by Filtration (Invitrogen)
Soluble fraction Insoluble fraction
MW,kDa
110 80
56
35 30
25
Purification of ProteinPurification of Protein
Scale up expression of successful constructs
or Purify protein from the soluble fraction
with Ni resin on the plateor
Purify protein from soluble fraction using AKTA autosampler
Efficiency of StepsEfficiency of Steps
PCR: 100% 94 out of 96 – first round ( robotic) 96 out of 96 – after manual repeating of 2 samplesCorrect plasmid: 96% 55 positive, 3 negative, 24 unclear, 14 no information –
after screening of 2 plates 92 positive, 4 no insert after screening of additional 2
platesExpression of protein (1 set of conditions): 51 – expression of protein, 34 – expression in soluble
fractionScale up of protein expression: 1 – soluble fraction
FinanceFinancestep chemicals Plate Tips accessories Total price PCR Oligo 20c/base
~$7/oligo Pfx polymerase ~$100/plate dNTP ~$20/plate
1PCR 3 Sealing – 1 PCR, 1 foil
1 $120 $350-700(oligo)
Agarose gel
Precast gel $20/gel
1 1 foil 1 $24
Clean up PCR
Ultrafiltration plate $33/plate
1 standard 2-3 1 scotch 1 $46
Digestion Enzymes ~$20/plate
1PCR 1 1foil 1 $29
Clean up digestion
Ultrafiltration plate $33/plate
1 standard 2-3 1 foil 1 $46
Ligation Ligase ~$100/plate
1PCR 2 1 foil 1 $113
Transformation
2 48-well
1 1 foil 1 $20
Plasmid purification
Qiagen Turbo kit $182/plate Eppendorf kit ~$70/plate
4 4 breathable 4 foil
4 $90x4=360
Plasmid digestion
Enzymes ~$20/plate
1PCR 1 1foil 4 $29x4=116
Agarose gel
Precast gel $20/gel
1 1 foil 4 $24x4=96
Transformation
2 48-well
1 1 foil 1 $20
Cell growth
1 standard 1 deepwell
2 1 foil, 1 breathable
1-6 $13/plate $13-78
Preparation soluble and insoluble fractions
Invitrogen kit ~$70/plate
1 2 foil 1-6 $75/plate $75-450
SDS-PAGE
$13/gel 8 gel /plate
2 PCR 4 2 foil 1-6 $130/plate 130-780
Ni-plate ~$65/plate 2 2 1 foil 1 $75 $2300.00
(70 oligo, 4 plasmid preps, 4 protein
$900.00+Priceofoligo