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Revision as of 15:03, 14 October 2018
EXPERIMENTS
Part I CRISPR
Part II CRISPR Confirmation (genotypic)
Part III: Designing of the new constructs (this is just modeling the construct design and not really a protocol)
Part IV: Gibson Assembly of Constructs
Part V: Deciding which fluorescent marker to use (water melon tubes, and plates)..deciding then to only use the GFP ones
Part VI: Genotypic and Phenotypic Proof of Concept
- a. Agarose gel electrophoresis of plasmid constructs (agarose gel electorphoresis protocol)
- b. Phenotypic Proof (Transformation and growing on selection plates)
Part VII: Quantifying Protein
- a. GFP protein purification using column chromatography
- b. Sialic Acid Assay - original version of protocol
Part VIII: Incorporation of New information to use His-tag
- a. Redesign construct to include His-tag
- b. Gibson assembly
- c. Transformation
- d. Protein Purification using His Tag Protein Chromatography -
redone Oct 10 (10 trials)
- c. Sialic Acid Purification redone using new protocol (6 trials)
- d. Fluoroscence Microplate Reading to Quantify Protein Concentration of Constructs
using GFP standard curve
- e. Fluorescence Microplate Reading to Quantify Sialic Acid Concentration
using Sialic Acid Standard Curve
- f. Final Polyacrylamide Gel Confirmation of Constructs
Part III: Designing of the new constructs (this is just modeling the construct design and not really a protocol)
Part IV: Gibson Assembly of Constructs
Part V: Deciding which fluorescent marker to use (water melon tubes, and plates)..deciding then to only use the GFP ones
Part VI: Genotypic and Phenotypic Proof of Concept
- a. Agarose gel electrophoresis of plasmid constructs (agarose gel electorphoresis protocol)
- b. Phenotypic Proof (Transformation and growing on selection plates)
Part VII: Quantifying Protein
- a. GFP protein purification using column chromatography
- b. Sialic Acid Assay - original version of protocol
Part VIII: Incorporation of New information to use His-tag
- a. Redesign construct to include His-tag
- b. Gibson assembly
- c. Transformation
- d. Protein Purification using His Tag Protein Chromatography -
redone Oct 10 (10 trials)
- c. Sialic Acid Purification redone using new protocol (6 trials)
- d. Fluoroscence Microplate Reading to Quantify Protein Concentration of Constructs
using GFP standard curve
- e. Fluorescence Microplate Reading to Quantify Sialic Acid Concentration
using Sialic Acid Standard Curve
- f. Final Polyacrylamide Gel Confirmation of Constructs
Part V: Deciding which fluorescent marker to use (water melon tubes, and plates)..deciding then to only use the GFP ones
Part VI: Genotypic and Phenotypic Proof of Concept
- a. Agarose gel electrophoresis of plasmid constructs (agarose gel electorphoresis protocol)
- b. Phenotypic Proof (Transformation and growing on selection plates)
Part VII: Quantifying Protein
- a. GFP protein purification using column chromatography
- b. Sialic Acid Assay - original version of protocol
Part VIII: Incorporation of New information to use His-tag
- a. Redesign construct to include His-tag
- b. Gibson assembly
- c. Transformation
- d. Protein Purification using His Tag Protein Chromatography -
redone Oct 10 (10 trials)
- c. Sialic Acid Purification redone using new protocol (6 trials)
- d. Fluoroscence Microplate Reading to Quantify Protein Concentration of Constructs
using GFP standard curve
- e. Fluorescence Microplate Reading to Quantify Sialic Acid Concentration
using Sialic Acid Standard Curve
- f. Final Polyacrylamide Gel Confirmation of Constructs
- a. Agarose gel electrophoresis of plasmid constructs (agarose gel electorphoresis protocol)
- b. Phenotypic Proof (Transformation and growing on selection plates)
Part VII: Quantifying Protein
- a. GFP protein purification using column chromatography
- b. Sialic Acid Assay - original version of protocol
Part VIII: Incorporation of New information to use His-tag
- a. Redesign construct to include His-tag
- b. Gibson assembly
- c. Transformation
- d. Protein Purification using His Tag Protein Chromatography -
redone Oct 10 (10 trials)
- c. Sialic Acid Purification redone using new protocol (6 trials)
- d. Fluoroscence Microplate Reading to Quantify Protein Concentration of Constructs
using GFP standard curve
- e. Fluorescence Microplate Reading to Quantify Sialic Acid Concentration
using Sialic Acid Standard Curve
- f. Final Polyacrylamide Gel Confirmation of Constructs
- a. Redesign construct to include His-tag
- b. Gibson assembly
- c. Transformation
- d. Protein Purification using His Tag Protein Chromatography - redone Oct 10 (10 trials)
- c. Sialic Acid Purification redone using new protocol (6 trials)
- d. Fluoroscence Microplate Reading to Quantify Protein Concentration of Constructs using GFP standard curve
- e. Fluorescence Microplate Reading to Quantify Sialic Acid Concentration using Sialic Acid Standard Curve
- f. Final Polyacrylamide Gel Confirmation of Constructs