Difference between revisions of "Team:Toronto/Experiments"

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{{Toronto}}
 
 
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href="https://2016.igem.org/Template:Toronto/Notebook/CSS?action=raw&ctype=text/css" />
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<div id="DIV_1">
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<div id="DIV_2">
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<p>Describe the experiments, research and protocols you used in your iGEM project.</p>
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<h1 id="H1_3">
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<div id="DIV_4">
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</div>
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6x Laemmli SDS Sample Loading Buffer
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</div>
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<div class="column half_size">
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</h1>
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<h5>What should this page contain?</h5>
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<div id="DIV_5">
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<ul>
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<h2 id="H2_6">
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<li> Protocols </li>
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Introduction
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<li> Experiments </li>
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</h2>
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<li>Documentation of the development of your project </li>
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<div id="DIV_7">
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</ul>
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<div id="DIV_8">
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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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</div>
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</div>
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</div>
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<div class="column half_size">
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<h2 id="H2_39">
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<h5>Inspiration</h5>
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Materials
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<ul>
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</h2>
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<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
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<div id="DIV_40">
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<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
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<div id="DIV_41">
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<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
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</div>
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</ul>
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<ul id="UL_42">
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</div>
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<li id="LI_43">
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<div id="DIV_44">
−
 
+
Reagents
−
<div class="clear"></div>
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</div>
−
 
+
<ul id="UL_45">
−
 
+
<li id="LI_46">
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<div class="column half_size">
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<div id="DIV_47">
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+
375mM Tris. HCL pH8.8
−
 
+
</div>
−
 
+
</li>
 +
<li id="LI_48">
 +
<div id="DIV_49">
 +
9%SDS
 +
</div>
 +
</li>
 +
<li id="LI_50">
 +
<div id="DIV_51">
 +
50% Glycerol
 +
</div>
 +
</li>
 +
<li id="LI_52">
 +
<div id="DIV_53">
 +
0.03% Bromophenol blue
 +
</div>
 +
</li>
 +
<li id="LI_54">
 +
<div id="DIV_55">
 +
<span id="SPAN_56"><span id="SPAN_57">14.7M β-mercaptoethanol</span></span>
 +
</div>
 +
</li>
 +
</ul>
 +
</li>
 +
<li id="LI_58">
 +
<div id="DIV_59">
 +
Equipment
 +
</div>
 +
<ul id="UL_60">
 +
<li id="LI_61">
 +
<div id="DIV_62">
 +
Micropipetter + tips
 +
</div>
 +
</li>
 +
<li id="LI_63">
 +
<div id="DIV_64">
 +
Beaker
 +
</div>
 +
</li>
 +
<li id="LI_65">
 +
<div id="DIV_66">
 +
Waterbath
 +
</div>
 +
</li>
 +
<li id="LI_67">
 +
<div id="DIV_68">
 +
microcentrifuge
 +
</div>
 +
</li>
 +
<li id="LI_69">
 +
<div id="DIV_70">
 +
microcentrifuge tube
 +
</div>
 +
</li>
 +
</ul>
 +
</li>
 +
</ul>
 +
</div>
 +
<h2 id="H2_71">
 +
Procedure
 +
</h2>
 +
<div id="DIV_72">
 +
<div id="DIV_73">
 +
<div id="DIV_74">
 +
</div>
 +
<div id="DIV_75">
 +
<ul id="UL_76">
 +
<li id="LI_77">
 +
<div id="DIV_78">
 +
<div id="DIV_79">
 +
Production of 100mL of stock
 +
</div><span id="SPAN_80"></span>
 +
</div>
 +
<div id="DIV_81">
 +
<div id="DIV_82">
 +
</div><span id="SPAN_83"></span>
 +
</div>
 +
</li>
 +
</ul>
 +
</div>
 +
<div id="DIV_84">
 +
<ol id="OL_85">
 +
<li id="LI_86">
 +
<div id="DIV_87">
 +
<div id="DIV_88">
 +
<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>
 +
</div><span id="SPAN_99"></span>
 +
</div>
 +
<div id="DIV_100">
 +
<div id="DIV_101">
 +
</div><span id="SPAN_102"></span>
 +
</div>
 +
</li>
 +
</ol>
 +
</div>
 +
<div id="DIV_103">
 +
<div id="DIV_104">
 +
<div id="DIV_105">
 +
<noscript id="NOSCRIPT_106">
 +
</noscript>
 +
<noscript id="NOSCRIPT_107">
 +
</noscript>
 +
<noscript id="NOSCRIPT_108">
 +
</noscript>
 +
<noscript id="NOSCRIPT_109">
 +
</noscript>
 +
</div>
 +
<div id="DIV_110">
 +
</div>
 +
<div id="DIV_111">
 +
</div>
 +
<div id="DIV_112">
 +
<div id="DIV_113">
 +
<div id="DIV_114">
 +
</div>
 +
<table id="TABLE_115">
 +
<colgroup id="COLGROUP_116">
 +
<col id="COL_117" />
 +
<col id="COL_118" />
 +
<col id="COL_119" />
 +
</colgroup>
 +
<tbody id="TBODY_120">
 +
<tr id="TR_121">
 +
<td id="TD_122">
 +
<span id="SPAN_123"></span>
 +
</td>
 +
<td id="TD_124">
 +
<div id="DIV_125">
 +
<span id="SPAN_126">A</span><span id="SPAN_127"><i id="I_128"></i></span>
 +
</div>
 +
</td>
 +
<td id="TD_129">
 +
<div id="DIV_130">
 +
<span id="SPAN_131">B</span><span id="SPAN_132"><i id="I_133"></i></span>
 +
</div>
 +
</td>
 +
</tr>
 +
<tr id="TR_134">
 +
<td id="TD_135">
 +
<div id="DIV_136">
 +
<span id="SPAN_137">1</span><span id="SPAN_138"><i id="I_139"></i></span>
 +
</div>
 +
</td>
 +
<td id="TD_140">
 +
<span id="SPAN_141">Reagent</span>
 +
</td>
 +
<td id="TD_142">
 +
<span id="SPAN_143">Quantity</span>
 +
</td>
 +
</tr>
 +
<tr id="TR_144">
 +
<td id="TD_145">
 +
<div id="DIV_146">
 +
<span id="SPAN_147">2</span><span id="SPAN_148"><i id="I_149"></i></span>
 +
</div>
 +
</td>
 +
<td id="TD_150">
 +
<span id="SPAN_151">375mM Tris-HCl</span>
 +
</td>
 +
<td id="TD_152">
 +
<span id="SPAN_153">5.91 g</span>
 +
</td>
 +
</tr>
 +
<tr id="TR_154">
 +
<td id="TD_155">
 +
<div id="DIV_156">
 +
<span id="SPAN_157">3</span><span id="SPAN_158"><i id="I_159"></i></span>
 +
</div>
 +
</td>
 +
<td id="TD_160">
 +
<span id="SPAN_161">9% SDS</span>
 +
</td>
 +
<td id="TD_162">
 +
<span id="SPAN_163">6 g</span>
 +
</td>
 +
</tr>
 +
<tr id="TR_164">
 +
<td id="TD_165">
 +
<div id="DIV_166">
 +
<span id="SPAN_167">4</span><span id="SPAN_168"><i id="I_169"></i></span>
 +
</div>
 +
</td>
 +
<td id="TD_170">
 +
<span id="SPAN_171">50% Glycerol</span>
 +
</td>
 +
<td id="TD_172">
 +
<span id="SPAN_173">48 mL</span>
 +
</td>
 +
</tr>
 +
<tr id="TR_174">
 +
<td id="TD_175">
 +
<div id="DIV_176">
 +
<span id="SPAN_177">5</span><span id="SPAN_178"><i id="I_179"></i></span>
 +
</div>
 +
</td>
 +
<td id="TD_180">
 +
<span id="SPAN_181">0.03% Bromophenol blue</span>
 +
</td>
 +
<td id="TD_182">
 +
<span id="SPAN_183">30mg</span>
 +
</td>
 +
</tr>
 +
<tr id="TR_184">
 +
<td id="TD_185">
 +
<div id="DIV_186">
 +
<span id="SPAN_187">6</span><span id="SPAN_188"><i id="I_189"></i></span>
 +
</div>
 +
</td>
 +
<td id="TD_190">
 +
<span id="SPAN_191">MilliQ (ddH2O)</span>
 +
</td>
 +
<td id="TD_192">
 +
<span id="SPAN_193">top to bring total to 100mL</span>
 +
</td>
 +
</tr>
 +
<tr id="TR_194">
 +
<td id="TD_195">
 +
<i id="I_196"></i>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<div id="DIV_197">
 +
<div id="DIV_198">
 +
</div>
 +
</div>
 +
</div>
 +
</div>
 +
<div id="DIV_199">
 +
<div id="DIV_200">
 +
<div id="DIV_201">
 +
Table1
 +
</div>
 +
<div id="DIV_202">
 +
</div>
 +
</div>
 +
</div>
 +
</div>
 +
</div>
 +
<div id="DIV_203">
 +
<ol id="OL_204">
 +
<li id="LI_205">
 +
<div id="DIV_206">
 +
<div id="DIV_207">
 +
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>
 +
</div><span id="SPAN_215"></span>
 +
</div>
 +
<div id="DIV_216">
 +
<div id="DIV_217">
 +
</div><span id="SPAN_218"></span>
 +
</div>
 +
</li>
 +
</ol>
 +
</div>
 +
<div id="DIV_219">
 +
<ol id="OL_220">
 +
<li id="LI_221">
 +
<div id="DIV_222">
 +
<div id="DIV_223">
 +
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.
 +
</div><span id="SPAN_225"></span>
 +
</div>
 +
<div id="DIV_226">
 +
<div id="DIV_227">
 +
</div><span id="SPAN_228"></span>
 +
</div>
 +
</li>
 +
</ol>
 +
</div>
 +
<div id="DIV_229">
 +
<ul id="UL_230">
 +
<li id="LI_231">
 +
<div id="DIV_232">
 +
<div id="DIV_233">
 +
Using 6X SDS Sample loading buffer
 +
</div><span id="SPAN_234"></span>
 +
</div>
 +
<div id="DIV_235">
 +
<div id="DIV_236">
 +
</div><span id="SPAN_237"></span>
 +
</div>
 +
</li>
 +
</ul>
 +
</div>
 +
<div id="DIV_238">
 +
<ol id="OL_239">
 +
<li id="LI_240">
 +
<div id="DIV_241">
 +
<div id="DIV_242">
 +
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>
 +
</div><span id="SPAN_247"></span>
 +
</div>
 +
<div id="DIV_248">
 +
<div id="DIV_249">
 +
</div><span id="SPAN_250"></span>
 +
</div>
 +
</li>
 +
</ol>
 +
</div>
 +
<div id="DIV_251">
 +
<ol id="OL_252">
 +
<li id="LI_253">
 +
<div id="DIV_254">
 +
<div id="DIV_255">
 +
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>
 +
</div><span id="SPAN_258"></span>
 +
</div>
 +
<div id="DIV_259">
 +
<div id="DIV_260">
 +
</div><span id="SPAN_261"></span>
 +
</div>
 +
</li>
 +
</ol>
 +
</div>
 +
<div id="DIV_262">
 +
<ul id="UL_263">
 +
<li id="LI_264">
 +
<div id="DIV_265">
 +
<div id="DIV_266">
 +
<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>
 +
</div><span id="SPAN_274"></span>
 +
</div>
 +
</li>
 +
</ul>
 +
</div>
 +
<div id="DIV_275">
 +
<ol id="OL_276">
 +
<li id="LI_277">
 +
<div id="DIV_278">
 +
<div id="DIV_279">
 +
Heat prepared protein sample at 100<sup id="SUP_280">o</sup>C for 5 minutes.
 +
</div><span id="SPAN_281"></span>
 +
</div>
 +
<div id="DIV_282">
 +
<div id="DIV_283">
 +
</div><span id="SPAN_284"></span>
 +
</div>
 +
</li>
 +
</ol>
 +
</div>
 +
<div id="DIV_285">
 +
<ol id="OL_286">
 +
<li id="LI_287">
 +
<div id="DIV_288">
 +
<div id="DIV_289">
 +
Breifly centrifuge heated sample and load into SDS polyacrylamide gel.
 +
</div><span id="SPAN_290"></span>
 +
</div>
 +
<div id="DIV_291">
 +
<div id="DIV_292">
 +
</div><span id="SPAN_293"></span>
 +
</div>
 +
</li>
 +
</ol>
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</div>
 +
<div id="DIV_294">
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<ul id="UL_295">
 +
<li id="LI_296">
 +
<div id="DIV_297">
 +
<div id="DIV_298">
 +
<i id="I_299">*****Note: B-mercaptoethanol rapidly oxidizes in protein loading buffer. Fresh 6X protein loading buffer shoudl be prepared every time!*****</i>
 +
</div><span id="SPAN_301"></span>
 +
</div>
 +
</li>
 +
</ul>
 +
</div>
 +
<div id="DIV_302">
 +
<ul id="UL_303">
 +
<li id="LI_304">
 +
<div id="DIV_305">
 +
<div id="DIV_306">
 +
Reference
 +
</div><span id="SPAN_307"></span>
 +
</div>
 +
<div id="DIV_308">
 +
<div id="DIV_309">
 +
</div><span id="SPAN_310"></span>
 +
</div>
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</li>
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</ul>
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</div>
 +
<div id="DIV_311">
 +
<ul id="UL_312">
 +
<li id="LI_313">
 +
<div id="DIV_314">
 +
<div id="DIV_315">
 +
http://www.wikiprotocols.org/protocols/formulation-of-6x-sds-sample-buffer/10090
 +
</div><span id="SPAN_316"></span>
 +
</div>
 +
</li>
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</ul>
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</div>
 +
<div id="DIV_317">
 +
<ul id="UL_318">
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<li id="LI_319">
 +
<div id="DIV_320">
 +
<div id="DIV_321">
 +
http://www.morganvillesci.com/6X-SDS-Protein-Loading-Buffer-25-mL-LB0100.htm
 +
</div><span id="SPAN_322"></span>
 +
</div>
 +
</li>
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</ul>
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</div>
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<div id="DIV_323">
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<ul id="UL_324">
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<li id="LI_325">
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<div id="DIV_326">
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<div id="DIV_327">
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Changelong
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</div><span id="SPAN_328"></span>
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</div>
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<div id="DIV_329">
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<div id="DIV_330">
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</div><span id="SPAN_331"></span>
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</div>
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</li>
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</ul>
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</div>
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<div id="DIV_332">
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<ul id="UL_333">
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<li id="LI_334">
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<div id="DIV_335">
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<div id="DIV_336">
 +
Created 5/17/2016
 +
</div><span id="SPAN_337"></span>
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</div>
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</li>
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</ul>
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</div>
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Revision as of 15:15, 27 September 2016

6x Laemmli SDS Sample Loading Buffer

Introduction

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) SAFETY PRECAUTIONS SDS (safety data sheet): 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. PPE (Personal protective equipment): 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. HAZARDS: β-mercaptoethanol: Combustible Liquid, Toxic by inhalation., Toxic by ingestion, Toxic by skin absorption, Moderate skin irritant, Severe eye irritant

Materials

  • Reagents
    • 375mM Tris. HCL pH8.8
    • 9%SDS
    • 50% Glycerol
    • 0.03% Bromophenol blue
    • 14.7M β-mercaptoethanol
  • Equipment
    • Micropipetter + tips
    • Beaker
    • Waterbath
    • microcentrifuge
    • microcentrifuge tube

Procedure

  • Production of 100mL of stock
  1. Add 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 ddH2O.
A
B
1
Reagent Quantity
2
375mM Tris-HCl 5.91 g
3
9% SDS 6 g
4
50% Glycerol 48 mL
5
0.03% Bromophenol blue 30mg
6
MilliQ (ddH2O) top to bring total to 100mL
Table1
  1. Add 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 ddH2O.
  1. Mix well and dissolve any percipitates in sample loading buffer by incubating at 37oC. This solution can be used as stock solution.
  • Using 6X SDS Sample loading buffer
  1. Add 9μL β-mercaptoethanol to 91 μLSDS Protein Loading Buffer and mix well. Invert 10 times.
  1. Make a 1:5 dilution of SDS Protein Loading Buffer (containing the reducing agent) to protein sample.
  • For example add 1μL of buffer to 5μL of sample protein.
  1. Heat prepared protein sample at 100oC for 5 minutes.
  1. Breifly centrifuge heated sample and load into SDS polyacrylamide gel.
  • *****Note: B-mercaptoethanol rapidly oxidizes in protein loading buffer. Fresh 6X protein loading buffer shoudl be prepared every time!*****
  • Reference
  • http://www.wikiprotocols.org/protocols/formulation-of-6x-sds-sample-buffer/10090
  • http://www.morganvillesci.com/6X-SDS-Protein-Loading-Buffer-25-mL-LB0100.htm
  • Changelong
  • Created 5/17/2016