Difference between revisions of "Team:Kent/Experiments"

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<label for="acc-close" class="hull-title">Complex Protocols</label>
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</header>
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<section class="hull">
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<label class="hull-title" for="cb15">Calibration of OD 600 Reference Point</label>
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<label class="hull-close" for="acc-close"></label>
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<div class="hull-content">
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LUDOX-S40 must be used as a single point reference with the aim to attain a
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ratiometric conversion factor, which in turn will be used to transform absorbance
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data into standard OD 600 measurement.
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<br>
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Standard 1 cm path length spectrophotometer readings are instrument dependent,
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while plate readers possess a path length less than 1 cm and are volume dependent.
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<br>
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Therefore, in this situation, there are 2 key objectives:
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<ul><li>Ratiometric conversion to transform Abs 600 measurements into OD 600
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measurements</li>
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<li> Accounting for instrument differences</li></ul>
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<br>
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Before starting the protocol, path length correction must be switched off. This is
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because scattering increases with longer wavelengths therefore adjustment is
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confounded by scattering solutions such as dense cells. However, many plate
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readers have automatic path length correction which is based on volume
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adjustment that uses ratio of absorbance measurements at 900 + 950 nm.
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LUDOX solution is only weakly scattering so will produce low absorbance values
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<br>
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Use same cuvettes, plates and volumes that are going to be used in cell based assays
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<br>
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Materials:
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<ul><li>1 mL 100% LUDOX</li>
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<li>H 2 O</li>
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<li>96 well plate (black with flat, transparent/clear bottom)</li></ul>
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<br>
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Method
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<ul><li>100 µl of LUDOX should be added into wells A1, B1, C1 and D2 (or 1mL into
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cuvette)</li>
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<li>100 µl of H 2 O should be added into wells A2, B2, C2 and D2</li>
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<li>Measure absorbance 600nm of all samples in all standard measurement modes in instrument</li>
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<li>Record data</li>
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</div>
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</section>
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<input type="radio" name="droptext" id="cb16" />
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<section class="hull">
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<label class="hull-title" for="cb16">Production of Fluorescein stock solution</label>
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<label class="hull-close" for="acc-close"></label>
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<div class="hull-content">
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<ul>
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<li> Spin down the Fluorescein stock tube and ensure the pellet is at the tubes' bottom </li>
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<li>Prepare 2x fluorescein stock solution (100 µM)<ul>
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<li>Resuspend Fluorescein in 1mL 1xPBS</li>
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<li>Ensure Fluorescein is properly dissolved<br>
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After the resuspension, pipette up and down and examine the solution in
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the tip (if particulates are visisble, continue to mix solution until they
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disappear)</li></ul></li>
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<br>
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<li>Dilute the 2x Fluorescein stock solution<ul>
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<li> With 1xPBS to make 1x fluorescein solution</li>
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<li>With resulting concentration of fluorescein stock solution 50 µM
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(500 µL of 2x fluorescein in 500 µL 1x PBS to make 1 mL of 50 µM (1x)
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fluorescein solution)</li></ul></li>
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</ul>
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</div>
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</section>
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<input type="radio" name="droptext" id="acc-close" />
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<input type="radio" name="droptext" id="cb17" />
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<section class="hull">
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<label class="hull-title" for="cb17">Fluorescein Fluorescence Standard Curve</label>
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<label class="hull-close" for="acc-close"></label>
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<div class="hull-content">
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A dilution series of Fluorescein in 4 replicates must be prepared where the
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fluorescence is measured in a 96 well plate in standard mode on a plate reader. A
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standard curve will be generated of fluorescence of fluorescein concentration. This
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will be used to correct cell based readings to an equivalent fluorescein
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concentration, which will then be converted into a GFP concentration.
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<br>
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<br>
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Materials
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<br>
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<ul><li>Fluorescein</li>
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<li>10mL 1xPBS (Phosphate Buffered Saline)</li>
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<li>96 well plate (black with flat, transparent/clear bottom)</li></ul>
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<br>
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Method
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<br>Serial dilutions need to be performed across columns 1-11
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Column 12 must contain PBS buffer only
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<br>
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The plate will initially be setup fluorescein stock in column 1 and equal volume of1xPBS in columns 2-12
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<ul><li> Add 100 µL of PBS into wells A2-A12, B2-B12, C2-C12 and D2-D12</li>
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<li>Add 200 µL of Fluorescein 1x stock solution into A1, B1, C1 and D1</li>
  
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<li>Transfer 100 µL of Fluorescein stock solution from A1 into A2</li>
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<li>Mix A2 by pipetting up and down 3x and transfer 100 µL into A3
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Repeat the process for A3 into A4, A4 into A5, etc. until A11</li>
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<li>Mix A11 by pipetting up and down 3x and transfer 100 µL into liquid waste</li>
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<li>Repeat dilution series for rows B, C and D</li>
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<li>Measure fluorescence of all samples in all standard measurement modes in
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instrument</li>
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<li>Record the data</li></ul>
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<br>
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Measurement notes
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<ul><li>The plates can now be measured in the plate reader</li>
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<li>Standard GFP settings must be used (same as those used when measuring the
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cells):<ul>
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<li>Excitation 485nm
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<li>Emission 530/30
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<li>Turn off path length correction</li></ul></li>
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<li>Would be ideal to repeat measurements with different settings
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<ul><li>Generates series of standard curves to choose from</li></ul></li>
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<li>Use number of settings that affect sensitivity (gain and/or slit width)
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<ul><li>Also consider orbital averaging, top/bottom optics</li></ul></li>
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</div>
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</section>
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<input type="radio" name="droptext" id="cb18" />
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<section class="hull">
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<label class="hull-title" for="cb18">Cell Measurement Protocol</label>
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<label class="hull-close" for="acc-close"></label>
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<div class="hull-content">
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The calibration measurements should be performed before the measurements on the cells are performed. This allows that the measurement process is understood
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and that the cell measurements are taken under the same conditions.
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<br>
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Materials
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<ul>
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<li>Competent cells (E.coli strain DH5-alpha)</li>
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<li>LB (Luria Bertani) media</li>
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<li>Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH –
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working stock 25 ug/mL)</li>
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<li>50 mL Falcon tube (covered in foil to block light)</li>
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<li>Incubator at 37oC</li>
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<li>1.5mL Eppendorf tubes for sample storage</li>
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<li>Ice bucket</li>
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<li>Pipettes</li>
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<li>96 well plate (black with flat, transparent/clear bottom)</li>************??????
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</ul>
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</div>
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</section>
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<input type="radio" name="droptext" id="cb19" />
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<section class="hull">
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<label class="hull-title" for="cb19">Calcium Chloride Competent Cells</label>
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<label class="hull-close" for="acc-close"></label>
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<div class="hull-content">
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Prior Preparation
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<ul><li>Autoclave 50mM Calcium Chloride and keep it cold at about 4 o C</li>
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<li>For the starter cultures<ul><li>
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<li>Add a colony of E.coli DH5cells to 5mL of LB</li>
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<li>Incubate at 37 o C overnight</li></ul></li>
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<br>
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Method:<ul>
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<li> Keep cells on ice at all times where possible</li>
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<li> To 100mLs of LB, add 100uL of cells from the overnight culture</li>
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<li> Let it grow at 37 o C and 250 rpm (until it reaches OD 600 ~0.6-0.8)</li>
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<li> Place cells on ice immediately to cool them once the correct OD 600 has been
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reached</li>
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<li>Centrifuge at max speed for 10 mins and 4 o C</li>
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<li>Discard supernatant</li>
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<li>Resuspend the pellet in 50% of the original volume with ice-cold 50mM CaCl 2; In a 5omL culture, add 25mL CaCl 2</li>
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<li>Allow them to sit on ice for 30 mins</li>
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<li>Centrifuge at max speed for 10 mins at 4 o C</li>
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<li>Discard the supernatant</li>
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</ul>
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<br>
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<div class="line-separator"></div>
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<br>
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Preparation of Competent Cells for Storage
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<br>
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<br>
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Materials
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<ul>
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<li>Cell Line</li>
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<li>Sterile LB</li>
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<li>10mM sterile and chilled Calcium Chloride</li>
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<li>Dry ice</li>
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<li>Acetone</li></ul>
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<br>
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Method
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<ul>
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<li>Inoculate the cells (either 1:50 or 1:100) into 50mL of LB</li>
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<li>Grow them at 37 o C until OD600 is around 0.4-0.5</li>
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<li>Place on ice for 10 minutes while Falcon tubes are pre-chilled</li>
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<li>The cells should be harvested at 3000 rpm, 4C for 8 minutes</li>
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<li>The pellet then needs to be resuspended in 1mL of 100mM CaCl 2 and 30%
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(v/v) glycerol</li>
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<li>The resulting solution needs to be aliquoted into chilled Eppendorf tubes
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(100uL per tube)</li>
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<li>Place each Eppendorf tube into an acetone dry ice bath to snap freeze them</li>
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<li>Then store at -80 o C</li></ul>
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</div>
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</section>
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<input type="radio" name="droptext" id="acc-close" />
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Revision as of 20:39, 29 October 2017


Experiments & Protocols