Team:Washington/Notebook

From 2009.igem.org

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= '''Protocols''' =
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A DETAILED DESCRIPTION OF THE PROTCOLS WE USED
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[[Team:Washington/Notebook/protein_gel|Protein Gel]]
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*Gene Synthesis
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[[Team:Washington/Notebook/Microscope|Microscopy]]
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*Colony PCR
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*Assembly
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*Cloning
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*Expression
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*Purification
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[[Team:Washington/Notebook/Flow_Cytometry|Flow Cytometry]]
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==== Protein Gel ====
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[[Team:Washington/Notebook/50mL_purification|Supernatant Protein Purification, 50mL]]
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# Set up overnights of parts 48-51
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# Dilute 1 ul overnight into 1ml
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# Add 1 mm IPTG and let grow for four hours
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# After cells have all grown up uniformly start boiling water for the boil step
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# Add 100uL of overnight to a 1.5mL tube
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# Pellet by spinning at max speed for 30 secs in the microcentrifuge
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# discard supernatent
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# Pull an aliquot of 5x sample loading buffer out of the freezer and thaw
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# Add 20uL BME to aliquot
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# Resuspend samples in 50uL sample loading buffer (pipette up/down)
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# Boil samples for 10 minutes
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# While boiling, prepare 500mL 1x SDS buffer:
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## 50mL 10x buffer to 450mL water
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## Take a gel out of the fridge and and put it in the gel box (keep the gel container for staining!!!)
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## Pour the mixed buffer solution into the half of the gel box that the gel is in
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## Remove the gel comb
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## Fill the little container on the top of the gel until it's about 0.5 cm from the top with buffer
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## Remove any bubbles in the wells
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# Spin down samples for a few seconds
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# Vortex samples
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# Load 3uL into each well
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# Load 10uL of ladder in appropriate wells
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# Run at 180V until the dye is about to fall off the gel
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==== Microscope ====
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[[Team:Washington/Notebook/Ni-column|Ni-column Set up]]
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# Set up overnights of parts 48-51. Let grow overnight.
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# Dilute 1 ul overnight into 1ml
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# Add 1 mm IPTG and let grow for four hours
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# After cells have grown up, place in flourophore (1 um) and allow time to bind (1 hour)
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# Also place 1 ul beads in 1 ml along with 1 ul flourophore.
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# Allow beads to bind to flourophore, then spin beads down, remove supernatent and replace with 1 ml water.
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# Next place the cells and the beads under the microscope
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==== Flow Cytometry ====
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[[Team:Washington/Notebook/2mL_purification|Supernatant Protein Purification, 2mL]]
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# Set up overnights of parts 48-51. Let grow overnight.
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# Dilute 1 ul overnight into 1ml
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# Add 1 mm IPTG and let grow for four hours
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# After cells have grown up, place in flourophore (1 um) and allow time to bind (1 hour)
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# Also place 1 ul beads in 1 ml along with 1 ul flourophore.
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# Allow beads to bind to flourophore, then spin beads down, remove supernatent and replace with 1 ml water.
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# Read the samples through the flow cytometer
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[[Team:Washington/Notebook/gene_synthesis|Gene Synthesis]]
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[[Team:Washington/Notebook/colony_PCR|Colony PCR]]
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[[Team:Washington/Notebook/NheI_PstI|BioBrick Assembly using the NheI and PstI sites]]
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[[Team:Washington/Notebook/NheI|BioBrick Assembly using the NheI site]]
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[[Team:Washington/Notebook/SOEingPCR|SOEing PCR]]
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[[Team:Washington/Notebook/IMAC_protocol|Traditional Protein Purification (IMAC)]]
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[[Team:Washington/Notebook/Standard_curve|Generating a Standard curve for GFP concentration]]
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<br>
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= '''Project Time Line''' =
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*Winter Quarter
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**Introduction to iGEM
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**Synthetic Biology Seminar
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**Plan for project ideas
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*Spring Quarter
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**Narrow down potential projects
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**Choose Project
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**Order oligos and start synthesizing genes
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**Obtained Funding
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**Stock Lab
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*June
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**Sequence genes
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**Preliminary binding assays for biotinylated fluorophore
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**Introduction to Fold-it as a tool for protein design
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**Test target proteins for solubility and expression
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**Start assembly of secretion genes
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*July
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**Develop and perform assays for testing legacy surface display bio-bricks
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**Transfer prtDEF contig from secretion system into low copy plasmid
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**Test target proteins for functionality
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*August
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**Finish assembly of secretion system
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**Start cell lines containing various forms of secretion system, make competent
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**Transform competent cells containing secretion system with target vector
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**Test for secretion of target protein
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**Start design of new display construct
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*September
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**Switch secretion system into new cell line, make cells competent
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**Transform with target vector
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**Test for secretion
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**Start Presentation
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**Start t-shirt design
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**Insert streptavidin into new display vector
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*October
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**Fine tune secretion assay, adjust controls
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**Finalize characterization of legacy parts
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**Practice presentation
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**Characterize target bio-bricks
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**Prepare for Jamboree
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A ROUGH TIMELINE OF THE PROJECT!
 
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Latest revision as of 20:20, 20 October 2009

Uw title logo.png

Protocols

Protein Gel

Microscopy

Flow Cytometry

Supernatant Protein Purification, 50mL

Ni-column Set up

Supernatant Protein Purification, 2mL

Gene Synthesis

Colony PCR

BioBrick Assembly using the NheI and PstI sites

BioBrick Assembly using the NheI site

SOEing PCR

Traditional Protein Purification (IMAC)

Generating a Standard curve for GFP concentration


Project Time Line

  • Winter Quarter
    • Introduction to iGEM
    • Synthetic Biology Seminar
    • Plan for project ideas


  • Spring Quarter
    • Narrow down potential projects
    • Choose Project
    • Order oligos and start synthesizing genes
    • Obtained Funding
    • Stock Lab


  • June
    • Sequence genes
    • Preliminary binding assays for biotinylated fluorophore
    • Introduction to Fold-it as a tool for protein design
    • Test target proteins for solubility and expression
    • Start assembly of secretion genes


  • July
    • Develop and perform assays for testing legacy surface display bio-bricks
    • Transfer prtDEF contig from secretion system into low copy plasmid
    • Test target proteins for functionality


  • August
    • Finish assembly of secretion system
    • Start cell lines containing various forms of secretion system, make competent
    • Transform competent cells containing secretion system with target vector
    • Test for secretion of target protein
    • Start design of new display construct


  • September
    • Switch secretion system into new cell line, make cells competent
    • Transform with target vector
    • Test for secretion
    • Start Presentation
    • Start t-shirt design
    • Insert streptavidin into new display vector


  • October
    • Fine tune secretion assay, adjust controls
    • Finalize characterization of legacy parts
    • Practice presentation
    • Characterize target bio-bricks
    • Prepare for Jamboree


Retrieved from "http://2009.igem.org/Team:Washington/Notebook"