Difference between revisions of "Team:Toronto/Experiments"

 
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<h1 id="H1_3">
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6x Laemmli SDS Sample Loading Buffer
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Introduction
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6x Protein Loading Buffer (Laemmli buffer) is used for the preparation of protein samples for SDS-poluacrylamide gel electrophoresis (SDS-PAGE). After the addition of the reducing agent β-mercaptoethanol (or DTT), the protein buffer will contain all of the necessary components for complete disruption of high-order protein structures. 6X Protein Loading Buffer is ideal because the protein sample prepared in 6X buffer will be more concentrated than protein sample prepared in 4X or 2X buffer (i.e. more protein and less loading buffer per well) <b id="B_9"><span id="SPAN_11"><span id="SPAN_12">SAFETY PRECAUTIONS</span></span></b> <span id="SPAN_13"><span id="SPAN_14"></span></span> <span id="SPAN_15"><span id="SPAN_16"></span></span> <b id="B_17"><span id="SPAN_18"><span id="SPAN_19">SDS (safety data sheet)</span></span></b><span id="SPAN_20"><span id="SPAN_21">: Refer to the SDS sheets for all listed materials before entering the lab. Be prepared to answer any questions regarding the information on these sheets.</span></span> <span id="SPAN_22"><span id="SPAN_23"></span></span> <span id="SPAN_24"><span id="SPAN_25"></span></span> <b id="B_26"><span id="SPAN_27"><span id="SPAN_28">PPE (Personal protective equipment):</span></span></b> <span id="SPAN_29"><span id="SPAN_30">Proper lab attire should be worn throughout the experiment: This means that upon entering the lab you should be wearing long pants and close-toed shoes. Contact lenses should not be worn. Furthermore, a lab coat, goggles, and gloves should be worn at all times, and long hair should be tied back.</span></span> <span id="SPAN_31"><span id="SPAN_32"></span></span> <b id="B_33"><span id="SPAN_35"><span id="SPAN_36">HAZARDS:</span></span></b> <span id="SPAN_37"><span id="SPAN_38">β-mercaptoethanol:</span></span> Combustible Liquid, Toxic by inhalation., Toxic by ingestion, Toxic by skin absorption, Moderate skin irritant, Severe eye irritant
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Materials
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Reagents
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375mM Tris. HCL pH8.8
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9%SDS
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50% Glycerol
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0.03% Bromophenol blue
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<span id="SPAN_56"><span id="SPAN_57">14.7M β-mercaptoethanol</span></span>
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Equipment
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Micropipetter + tips
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Beaker
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Waterbath
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microcentrifuge
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microcentrifuge tube
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Procedure
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Production of 100mL of stock
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<span id="SPAN_89"><span id="SPAN_90">Add</span></span> <span id="SPAN_91"><span id="SPAN_92">5.91 g Tris-HCl, 6 g SDS, 48 mL 100% glycerol, 9 mL 14.7 M 2-Mercaptoethanol, and 30 mg bromophenol blue. Bring to 100 mL with ddH</span></span><sub id="SUB_93"><span id="SPAN_94"><span id="SPAN_95"><span id="SPAN_96">2</span></span></span></sub><span id="SPAN_97"><span id="SPAN_98">O.</span></span>
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<span id="SPAN_126">A</span><span id="SPAN_127"><i id="I_128"></i></span>
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<span id="SPAN_141">Reagent</span>
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<span id="SPAN_143">Quantity</span>
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<span id="SPAN_151">375mM Tris-HCl</span>
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<span id="SPAN_153">5.91 g</span>
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<span id="SPAN_161">9% SDS</span>
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<span id="SPAN_163">6 g</span>
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<span id="SPAN_171">50% Glycerol</span>
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<span id="SPAN_173">48 mL</span>
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<span id="SPAN_181">0.03% Bromophenol blue</span>
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<span id="SPAN_183">30mg</span>
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<span id="SPAN_191">MilliQ (ddH2O)</span>
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<span id="SPAN_193">top to bring total to 100mL</span>
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Table1
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Add <span id="SPAN_208"><span id="SPAN_209">5.91 g Tris-HCl, 6 g SDS, 48 mL 100% glycerol, 9 mL 14.7 M 2-Mercaptoethanol, and 30 mg bromophenol blue. Bring to 100 mL with ddH</span></span><sub id="SUB_210"><span id="SPAN_211"><span id="SPAN_212">2</span></span></sub><span id="SPAN_213"><span id="SPAN_214">O.</span></span>
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Mix well and dissolve any percipitates in sample loading buffer by incubating at 37<sup id="SUP_224">o</sup>C. This solution can be used as stock solution.
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Using 6X SDS Sample loading buffer
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Add 9μL <span id="SPAN_243"><span id="SPAN_244">β-mercaptoethanol to 91</span></span> <span id="SPAN_245"><span id="SPAN_246">μLSDS Protein Loading Buffer and mix well. Invert 10 times.</span></span>
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Make a 1:5 dilution of <span id="SPAN_256"><span id="SPAN_257">SDS Protein Loading Buffer (containing the reducing agent) to protein sample.</span></span>
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<i id="I_267">For example add 1</i><span id="SPAN_268"><span id="SPAN_269">μL <i id="I_270">of buffer to 5</i></span></span><span id="SPAN_271"><span id="SPAN_272">μL <i id="I_273">of sample protein.</i></span></span>
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Heat prepared protein sample at 100<sup id="SUP_280">o</sup>C for 5 minutes.
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Breifly centrifuge heated sample and load into SDS polyacrylamide gel.
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<i id="I_299">*****Note: B-mercaptoethanol rapidly oxidizes in protein loading buffer. Fresh 6X protein loading buffer shoudl be prepared every time!*****</i>
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Reference
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http://www.wikiprotocols.org/protocols/formulation-of-6x-sds-sample-buffer/10090
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Created 5/17/2016
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(REVISE) RFP + LacI backbone PCR
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<div id="navigation">
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<ul>
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<li><a href="https://2016.igem.org/Team:Toronto"><span>home</span></a></li>
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</li>
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<li><a href="https://2016.igem.org/Team:Toronto/Achievements"><span>achievements</span></a></li>
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</li>
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<li><a href="#"><span>team</span></a>
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<ul>
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<li><a href="https://2016.igem.org/Team:Toronto/Team"><span>team</span></a></li>
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</li>
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<li><a href="https://2016.igem.org/Team:Toronto/Collaborations"><span>collaborations</span></a></li>
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</li>
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</ul>
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<li class="active"><a href="#"><span>project</span></a>
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<ul>
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<li><a href="https://2016.igem.org/Team:Toronto/Description"><span>description</span></a></li>
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</li>
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<li><a href="https://2016.igem.org/Team:Toronto/Design"><span>design</span></a></li>
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</li>
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<li class="active"><a href="https://2016.igem.org/Team:Toronto/Experiments"><span>experiments</span></a></li>
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</li>
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<li><a href="https://2016.igem.org/Team:Toronto/Proof"><span>proof</span></a></li>
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</li>
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<li><a href="https://2016.igem.org/Team:Toronto/Demonstrate"><span>demonstrate</span></a></li>
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</li>
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<li><a href="https://2016.igem.org/Team:Toronto/Results"><span>results</span></a></li>
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</li>
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<li><a href="https://2016.igem.org/Team:Toronto/Notebook"><span>notebook</span></a></li>
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</li>
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</ul>
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<li><a href="#"><span>parts</span></a>
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<ul>
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<li><a href="https://2016.igem.org/Team:Toronto/Parts"><span>parts</span></a></li>
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</li>
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<li><a href="https://2016.igem.org/Team:Toronto/Basic_Part"><span>basic_part</span></a></li>
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</li>
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<li><a href="https://2016.igem.org/Team:Toronto/Composite_Part"><span>composite_part</span></a></li>
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</li>
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<li><a href="https://2016.igem.org/Team:Toronto/Part_Collection"><span>part_collection</span></a></li>
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</li>
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</ul>
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<li><a href="https://2016.igem.org/Team:Toronto/Safety"><span>safety</span></a></li>
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</li>
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<li><a href="https://2016.igem.org/Team:Toronto/Attributions"><span>attributions</span></a></li>
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</li>
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<li><a href="#"><span>human_practices</span></a>
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<ul>
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<li><a href="https://2016.igem.org/Team:Toronto/Human_Practices"><span>human_practices</span></a></li>
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</li>
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<li><a href="https://2016.igem.org/Team:Toronto/HP-Silver"><span>silver</span></a></li>
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</li>
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<li><a href="https://2016.igem.org/Team:Toronto/HP-Gold"><span>gold</span></a></li>
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</li>
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<li><a href="https://2016.igem.org/Team:Toronto/Integrated_Practices"><span>integrated_practices</span></a></li>
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</li>
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</ul>
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<li><a href="#"><span>awards</span></a>
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<ul>
 +
<li><a href="https://2016.igem.org/Team:Toronto/Software"><span>software</span></a></li>
 +
</li>
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<li><a href="https://2016.igem.org/Team:Toronto/Model"><span>model</span></a></li>
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</li>
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</ul>
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</ul>
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</div>
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<div class="content">
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<div class="content" id="content-main"><div class="row"><div class="col col-lg-8 col-md-12"><div class="content-main"><h1 id="experiments">Experiments</h1>
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<ul>
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<li><a href="https://2016.igem.org/Team:Toronto/Experiment-Bacterial_Transformation_Protocol">Bacterial Transformation Protocol</a></li>
 +
<li><a href="https://2016.igem.org/Team:Toronto/Experiment-Ethanol_70">Ethanol 70%</a></li>
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<li><a href="https://2016.igem.org/Team:Toronto/Experiment-Gel_Electrophoresis">Gel Electrophoresis</a></li>
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<li><a href="https://2016.igem.org/Team:Toronto/Experiment-Gel_Extraction">Gel Extraction</a></li>
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<li><a href="https://2016.igem.org/Team:Toronto/Experiment-Glycerol_Stock_Protocol_(Bacteria">Glycerol Stock Protocol (Bacteria)</a>)</li>
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<li><a href="https://2016.igem.org/Team:Toronto/Experiment-LB_media_preperation_(500mL;25_plates">LB media preperation (500mL;25 plates)</a>)</li>
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<li><a href="https://2016.igem.org/Team:Toronto/Experiment-Making_Chemically_Competent_Cells_(Openwetware">Making Chemically Competent Cells (Openwetware)</a>)</li>
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<li><a href="https://2016.igem.org/Team:Toronto/Experiment-Making_Electrocompetent_cells">Making Electrocompetent cells</a></li>
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<li><a href="https://2016.igem.org/Team:Toronto/Experiment-Monarch_Miniprep_Protocol">Monarch Miniprep Protocol</a></li>
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<li><a href="https://2016.igem.org/Team:Toronto/Experiment-Nanodrop">Nanodrop</a></li>
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<li><a href="https://2016.igem.org/Team:Toronto/Experiment-PureLink_PCR_Purification_protocol">PureLink PCR Purification protocol</a></li>
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<li><a href="https://2016.igem.org/Team:Toronto/Experiment-Quantum_Prep_Plasmid_Miniprep">Quantum Prep Plasmid Miniprep</a></li>
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<li><a href="https://2016.igem.org/Team:Toronto/Experiment-Restriction_Enzyme_Digest_and_Ligation">Restriction Enzyme Digest and Ligation</a></li>
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<li><a href="https://2016.igem.org/Team:Toronto/Experiment-Rubidium_Chloride_Competent_Cells">Rubidium Chloride Competent Cells</a></li>
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<li><a href="https://2016.igem.org/Team:Toronto/Experiment-S30_Protocol">S30 Protocol</a></li>
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<li><a href="https://2016.igem.org/Team:Toronto/Experiment-SOC_Medium_Protocol">SOC Medium Protocol</a></li>
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<li><a href="https://2016.igem.org/Team:Toronto/Experiment-TB_(Terrific_Broth)_Buffer">TB (Terrific Broth) Buffer</a></li>
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<li><a href="https://2016.igem.org/Team:Toronto/Experiment-Transformation_Efficiency">Transformation Efficiency</a></li>
 +
<li><a href="https://2016.igem.org/Team:Toronto/Experiment-iGEM-Competent-Cell-Test-Kit">iGEM Competent Cell Test Kit</a></li>
 +
<li><a href="https://2016.igem.org/Team:Toronto/Experiment-iGEM_DNA_Kit_Plate_Instructions">iGEM DNA Kit Plate Instructions</a></li>
 +
</ul>
 +
</div></div><div id="tableofcontents" class="tableofcontents affix sidebar col-lg-4 hidden-xs hidden-sm hidden-md visible-lg-3"><ul class="nav"></div></div></div>
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{{Toronto/footer}}

Latest revision as of 17:51, 19 October 2016

Experiments