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Overview of Bindley Bioscience Center Protein Production Lab: experimental capabilities.

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?

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Overview of Bindley Bioscience Center Protein Production Lab: experimental capabilities.

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  1. 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

  2. PCR plate Filtration plate Deepwell plate 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! Standard

  3. 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 instrument 2 cooling-heating modules, Waste disposal module.

  4. 2. MJResearch PCR Thermocycler “DNA Engine DYAD” (gradient available). 3. Sigma-Quiagen Centrifuge 4K15C with rotor for 96-well plates.

  5. 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. 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 7. Multichannel pipettors. “Matrix” “Biohit” min 0.2ul max 1200ul Max 1250ul min 1ul

  7. 8. Thermomixer, shaker, heated/refrigerated incubator with holders for microtiter plates. Vortex Benchtop Model Shaker-Incubator “Innova 4230” Rocking platform Orbital shaker Thermomixer

  8. Applications (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 to discuss your options.

  9. Design 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

  10. Insert preparation Primer design PCR fragment generation Digestion with cloning enzymes Standard Steps for Cloning Experiment Target choosing Vector preparation • Isolation of plasmid vector • Digestion with cloning enzymes 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

  11. PCR Amplification Bacterial transformation Invitrogen, Precast 1% agarose gel ~1500bp ~500bp • Chemical • Heat-shock Q-tray

  12. Procedure for plasmid purification 96 x 1–1.25 ml E. coli bacterial culture in 96-Deepwell Culture Plate Centrifuge 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 Filter Plate DB Place Filter Plate DB on top of Collection Plate

  13. Protein expression

  14. Separate phases by Filtration (Invitrogen) Screening for Soluble Protein MW, kDa 110 80 56 35 30 25 Soluble fraction Insoluble fraction

  15. Purification of Protein • Scale up expression of successful constructs or • Purify protein from the soluble fraction with Ni resin on the plate or • Purify protein from soluble fraction using AKTA autosampler

  16. Efficiency of Steps PCR: 100% • 94 out of 96 – first round ( robotic) • 96 out of 96 – after manual repeating of 2 samples Correct 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 plates Expression of protein (1 set of conditions): • 51 – expression of protein, 34 – expression in soluble fraction Scale up of protein expression: • 1 – soluble fraction

  17. Finance $900.00 + Price of oligo

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