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