Team:Washington/Protocols

From 2013.igem.org

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<b><u>Cloning Protocols Workflow</u></b>
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<b><u>Cloning Protocols</u></b>
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<b>Workflow:</b>
 
<ul>
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<li><a href = "https://2013.igem.org/Team:Washington/iGEMVectors">iGEM Vector Information</a></li>
<li><a href = "https://2013.igem.org/Team:Washington/iGEMVectors">iGEM Vector Information</a></li>
<li><a href = "https://2013.igem.org/Team:Washington/General_Cloning_Strategy">General Cloning Strategy</a></li>
<li><a href = "https://2013.igem.org/Team:Washington/General_Cloning_Strategy">General Cloning Strategy</a></li>
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<b>Workflow:</b>
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<ul>
<li><a href = "https://2013.igem.org/Team:Washington/ISOLATION_OF_PLASMID">1) Isolation of Plasmid DNA (miniprep)</a></li>
<li><a href = "https://2013.igem.org/Team:Washington/ISOLATION_OF_PLASMID">1) Isolation of Plasmid DNA (miniprep)</a></li>
<li><a href = "https://2013.igem.org/Team:Washington/GENERAL_PCR_PROTOCOL">2) General PCR Protocol </a>(also see <a href = "https://2013.igem.org/Team:Washington/PCR_GOTAG">PCR GoTaq</a> - product (30-200ng/ ul) Check on gel PCR Purification and/or  <a href = "https://2013.igem.org/Team:Washington/DPNL_DIGEST">Dpnl Digest</a></li>
<li><a href = "https://2013.igem.org/Team:Washington/GENERAL_PCR_PROTOCOL">2) General PCR Protocol </a>(also see <a href = "https://2013.igem.org/Team:Washington/PCR_GOTAG">PCR GoTaq</a> - product (30-200ng/ ul) Check on gel PCR Purification and/or  <a href = "https://2013.igem.org/Team:Washington/DPNL_DIGEST">Dpnl Digest</a></li>
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<li><a href = "https://2013.igem.org/Team:Washington/HEAT_SHOCK_CHEM_TRANS">6) Heat shock/chemical competent transformation</a></li>
<li><a href = "https://2013.igem.org/Team:Washington/HEAT_SHOCK_CHEM_TRANS">6) Heat shock/chemical competent transformation</a></li>
<li><a href = "https://2013.igem.org/Team:Washington/COLONY_PCR_GREEN">7) Colony PCR with Green taq</a> Miniprep (stocks can be made from this culture - add 1ml extra)</li>
<li><a href = "https://2013.igem.org/Team:Washington/COLONY_PCR_GREEN">7) Colony PCR with Green taq</a> Miniprep (stocks can be made from this culture - add 1ml extra)</li>
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<li><a href = "https://2013.igem.org/Team:Washington/DNA_SEQUENCING">8) DNA Sequencing</a></li>
 
<li><a href = "https://2013.igem.org/Team:Washington/MAKING_GLYCEROL_FROZEN_STOCKS">9) Making Glycerol Frozen Stocks</a></li>
<li><a href = "https://2013.igem.org/Team:Washington/MAKING_GLYCEROL_FROZEN_STOCKS">9) Making Glycerol Frozen Stocks</a></li>
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<b><u>Light Sensor Protocols</u></b>
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<b><u>Light Sensing Protocols</u></b>
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To carry out a typical Green light inducible GFP experiment:
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<b>Light Sensing Media:</b>
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1. Transform plasmids containing pLPCB and pJT119B operon to a competent cell. Our lab uses JT2 (Tabor, J. J. et al., Multichromatic Control of Gene Expression in Escherichia coli, J. Mol. Biol. (2010), doi:10.1016/j.jmb.2010.10.03) cells for gfp output testing.
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<ul><li><a href = "https://2013.igem.org/Team:Washington/M9Salts">5X M9 Salts</a></li>
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<li><a href = "https://2013.igem.org/Team:Washington/M9Media">M9 Media + Casamino Acid Recipe</a></li>
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2. Plate cells on LB agar plates with XXX and XXX antibiotics. See table below for list of systems and plasmids with their required antibiotics.
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<li><a href = "https://2013.igem.org/Team:Washington/M9Plate">M9 + Casamino Acid Plate</a></li></ul>
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<b>Light Testing:</b><ul>
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3. After incubation at 37 oC overnight, pick a single colony, and inoculate 5 mL of LB medium in a 50 mL Falcon tube.
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<li><a href = "https://2013.igem.org/Team:Washington/GeneralProtocol">General Protocol</a></li>
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<li><a href = "https://2013.igem.org/Team:Washington/96WellPlateAssay">96-Well Plate Assay</a></li>
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4. Grow cultures to saturation overnight at 37 oC with shaking.
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<li><a href = "https://2013.igem.org/Team:Washington/60mmPetriDishAssay">60 mm Petri Dish Assay</a></li>
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<li><a href = "https://2013.igem.org/Team:Washington/LightIntensityTest">Light Intensity Test</a></li>
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5. Determine cell density (O.D.600 measurement), and dilute overnight cultures to a final O.D.600 of 1x10-4 in M9 minimal medium supplemented with casamino acids
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<li><a href = "https://2013.igem.org/Team:Washington/BlinkingTest">Blinking Test</a></li>
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<li><a href = "https://2013.igem.org/Team:Washington/FluorescentAnalysisProtocol">Fluorescent Analysis Protocol</a></li>
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(link: http://openwetware.org/wiki/M9_salts 5x m9salts)
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<li><a href = "https://2013.igem.org/Team:Washington/GFPExpression">GFP Expression on Agar Plates</a></li>
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(link: http://openwetware.org/wiki/Endy:M9_media/supplemented m9 supplemented)and appropriate antibiotics.
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<b>Setting Up The App:</b><ul>
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<li><a href = "https://2013.igem.org/Team:Washington/appsetup">E.colight Setup</a></li></ul>
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For 96 well plates:
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<b>App Testing:</b><ul>
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<li><a href = "https://2013.igem.org/Team:Washington/BleedThroughTest">Bleed-Through Test</a></li>
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1.  Pipette 260 ul of the inoculated M9 + Casamino acid media
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(link: http://openwetware.org/wiki/M9_salts 5x m9salts)
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(link: http://openwetware.org/wiki/Endy:M9_media/supplemented into each well of interest in a clear bottomed black 96 well plate (Supplier information -- maybe even the name).
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2. Cover the plate with breathable tape.
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3. Orient the 96 well plate on the tablet such that the wells line up with the appropriate light source on the the tablet.
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4. Secure the 96 well plate on top of the tablet with tape.  
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5. Incubate tablet and 96 well plate on an orbital shaker (RPM?) for 8 hours at 37 oC.
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6. Remove 200 ul of culture from each well, and pipette them to 96 well plates for analysis (Highlight analysis and link to pertinent protocol).
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96 well analysis (GFP expression):
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1. GFP expression levels are analyzed on a spectramax m5e plate reader (bottom read) with an excitation of 480 nm, and emission at 520nm. Set the plate reader to automix the samples for 5 seconds prior to analysis.
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2. GFP expression levels were normalized against cell density by dividing the relative fluorescence unit values by O.D.600 data.
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For 60mm mini petridish:
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1. Add 7.5ml of the inoculatted M9+cassamino acid media into the plate which is labelled for green light testing.
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2. Add another 7.5ml of the inoculated media and a put it into another plate which is labelled as dark for the dark condition test.
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3. Foil the mini petri dish for the dark ones.
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4. Get another clear 60mm mini petri dish and then use it as a spacer plate from the tablet to the testing petri dish which will sit on top of the green light. This will prevent overheating of the culture. (refer to supplemental image)
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5. Tape both spacer and testing petri dish ontop of the testing tablet securely and put them on top of the orbital shaker in the 37C incubator.
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6. After eight hours, take 200ul of the culture which has been growing on top of the shaker and pipette them to 96well plates for RFU plate reading
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7. We took the Optical density of the cells with lambda 600nm
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8. Also took endpoint fluorescence which is excitation= 480nm, emission= 520nm, autocutoff= 515nm. Automixed 5seconds before reading.
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9. Then we normalized RFU(relative fluorescence unit) with the optical density by dividing the RFU with the OD.
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Latest revision as of 21:55, 27 September 2013



Cloning Protocols

Workflow:


Light Sensing Protocols

Light Sensing Media:

Light Testing: Setting Up The App: App Testing:

Retrieved from "http://2013.igem.org/Team:Washington/Protocols"