Difference between revisions of "Team:Bielefeld-CeBiTec/Media"
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<html> | <html> | ||
+ | |||
+ | <head> | ||
+ | <script> | ||
+ | $("#notebook").addClass("navbar-active"); | ||
+ | $(document).ready(function(){ | ||
+ | $("#button1").click(function(){ | ||
+ | $(".panel-collapse").collapse("show"); | ||
+ | }); | ||
+ | }); | ||
+ | $(document).ready(function(){ | ||
+ | $("#button2").click(function(){ | ||
+ | $(".panel-collapse").collapse("hide"); | ||
+ | }); | ||
+ | $(document).ready(function() { | ||
+ | var anchor = window.location.hash.replace("#", ""); | ||
+ | $(".collapse").collapse('hide'); | ||
+ | $("#" + anchor).collapse('show'); | ||
+ | }); | ||
+ | }); | ||
+ | </script> | ||
+ | </head> | ||
<body> | <body> | ||
<div class="container"> | <div class="container"> | ||
− | + | ||
− | + | <div class="jumbotron" style="background-image: url(https://static.igem.org/mediawiki/2015/b/b9/Bielefeld-CeBiTec-buffers.jpg)"> | |
+ | <div class="jumbotron-text"> | ||
+ | <h1 style="margin-bottom: 0px">Media & Buffers</h1> | ||
+ | <p>All of our favorite recipes.</p> | ||
</div> | </div> | ||
− | <div class="col-md-8"> | + | </div> |
+ | |||
+ | |||
+ | <div class="col-md-8 col-md-offset-2"> | ||
<h1>Media</h1> | <h1>Media</h1> | ||
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<li>For 1 liter LB:</li> | <li>For 1 liter LB:</li> | ||
<ul> | <ul> | ||
− | <li>20 g LB powder</li> | + | <li>20 g LB powder (Merck)</li> |
<li>Fill the bottle with deionized H<sub>2</sub>O</li><br> | <li>Fill the bottle with deionized H<sub>2</sub>O</li><br> | ||
</ul> | </ul> | ||
− | <li>For 1 | + | <li>For 1 L LB-plates:</li> |
<ul> | <ul> | ||
− | <li>18 g LB powder</li> | + | <li>18 g LB powder (Merck)</li> |
− | <li>16 g Select Agar</li> | + | <li>16 g Select Agar (Invitrogen)</li> |
<li>Fill the bottle with deionized H<sub>2</sub>O</li> | <li>Fill the bottle with deionized H<sub>2</sub>O</li> | ||
</ul> | </ul> | ||
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<div class="panel-body"> | <div class="panel-body"> | ||
<ul> | <ul> | ||
− | <li>Add the following components for 900 | + | <li>Add the following components for 900 mL of distilled H<sub>2</sub>O: </li> |
<ul> | <ul> | ||
<li>20 g Trypton</li> | <li>20 g Trypton</li> | ||
<li>5 g Bacto Yeast Extract </li> | <li>5 g Bacto Yeast Extract </li> | ||
− | <li>2 | + | <li>2 mL of 5 M NaCl </li> |
− | <li>2.5 | + | <li>2.5 mL of 1 M KCl </li> |
− | <li>10 | + | <li>10 mL of 1 M MgMgCl<sub>2</sub> </li> |
− | <li>10 | + | <li>10 mL of 1 M MgSO<sub>4</sub> </li> |
− | <li>20 | + | <li>20 mL of 1 M glucose </li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
Line 62: | Line 89: | ||
</div> | </div> | ||
</div> | </div> | ||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#collapseThree">2xYT+P</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="collapseThree" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li> For 1 L, combine </li> | ||
+ | <ul> | ||
+ | <li> 16 g tryptone</li> | ||
+ | <li> 10 g yeast extract</li> | ||
+ | <li> 5 g NaCl </li> | ||
+ | <li> 22 mL KH<sub>2</sub>PO<sub>4</sub></li> | ||
+ | <li> 40 mL K<sub>2</sub>HPO<sub>4</sub></li> | ||
+ | |||
+ | </ul> | ||
+ | <li>dissolve in water and titrate to pH = 7.0 with NaOH, autoclave </li> | ||
+ | |||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
</div> | </div> | ||
+ | |||
+ | |||
+ | </div> | ||
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<div class="panel-heading"> | <div class="panel-heading"> | ||
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a data-toggle="collapse" href="# | + | <a data-toggle="collapse" href="#collapseFour">50X TAE Stock Solution</a> |
</h4> | </h4> | ||
</div> | </div> | ||
− | <div id=" | + | <div id="collapseFour" class="panel-collapse collapse"> |
<div class="panel-body"> | <div class="panel-body"> | ||
<ul> | <ul> | ||
<li>For each litre of solution: </li> | <li>For each litre of solution: </li> | ||
<ul> | <ul> | ||
− | <li>242 g | + | <li>242 g TRIS Base (MW=121.1) </li> |
<li>57.1 ml Glacial Acetic Acid </li> | <li>57.1 ml Glacial Acetic Acid </li> | ||
<li>100 ml 0.5 M EDTA </li> | <li>100 ml 0.5 M EDTA </li> | ||
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<div class="panel-heading"> | <div class="panel-heading"> | ||
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a data-toggle="collapse" href="# | + | <a data-toggle="collapse" href="#collapseFive">Lysis and binding buffer for purification of LacI</a> |
</h4> | </h4> | ||
</div> | </div> | ||
− | <div id=" | + | <div id="collapseFive" class="panel-collapse collapse"> |
<div class="panel-body"> | <div class="panel-body"> | ||
<ul> | <ul> | ||
Line 108: | Line 162: | ||
<li>5.844 g NaCl </li> | <li>5.844 g NaCl </li> | ||
<li>0.272 g imidazole </li> | <li>0.272 g imidazole </li> | ||
− | <li>5.8 ml of 86 % glycerol </li> | + | <li>5.8 ml of 86 % (v/v) glycerol </li> |
<li>0.406 g MgCl<sub>2</sub></li> | <li>0.406 g MgCl<sub>2</sub></li> | ||
<li>0.2 g lysozyme</li> | <li>0.2 g lysozyme</li> | ||
</ul> <br> | </ul> <br> | ||
− | <li>Add all these ingredients to a 50 mM sodium phosphate buffer (pH 8.0). Prior to use add DTT to a final concentration of | + | <li>Add all these ingredients to a 50 mM sodium phosphate buffer (pH 8.0). Prior to use add DTT to a final concentration of 1 mM and tween 20 to a final concentration of 0.1 % (v/v). </li> |
<li>store at room temperature. </li> <br> | <li>store at room temperature. </li> <br> | ||
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<li>500 mM NaCl </li> | <li>500 mM NaCl </li> | ||
<li>20 mM imidazole </li> | <li>20 mM imidazole </li> | ||
− | <li>2.5 % | + | <li>2.5 % glycerol </li> |
<li>1 mM DTT </li> | <li>1 mM DTT </li> | ||
<li>10 mM MgCl<sub>2</sub> </li> | <li>10 mM MgCl<sub>2</sub> </li> | ||
− | <li>0.1 % tween 20 </li> | + | <li>0.1 % (v/v) tween 20 </li> |
− | <li>1 mg/ | + | <li>1 mg/mL lysozyme</li> |
</ul> | </ul> | ||
</ul> | </ul> | ||
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<div class="panel-heading"> | <div class="panel-heading"> | ||
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a data-toggle="collapse" href="# | + | <a data-toggle="collapse" href="#collapseSix">Equilibration buffer for LacI purification</a> |
</h4> | </h4> | ||
</div> | </div> | ||
− | <div id=" | + | <div id="collapseSix" class="panel-collapse collapse"> |
<div class="panel-body"> | <div class="panel-body"> | ||
<ul> | <ul> | ||
<li>For each litre of solution: </li> | <li>For each litre of solution: </li> | ||
<ul> | <ul> | ||
− | <li>29. | + | <li>29.22 g NaCl </li> |
<li>1.36 g imidazole </li> | <li>1.36 g imidazole </li> | ||
− | <li>29 ml of 86 % glycerol </li> | + | <li>29 ml of 86 % (v/v) glycerol </li> |
</ul> <br> | </ul> <br> | ||
− | <li>Add all these ingredients to a 50 mM sodium phosphate buffer (pH 8.0). Prior to use add DTT to a final concentration of | + | <li>Add all these ingredients to a 50 mM sodium phosphate buffer (pH 8.0). Prior to use add DTT to a final concentration of 1 mM. </li> |
<li>store at room temperature. </li> <br> | <li>store at room temperature. </li> <br> | ||
Line 153: | Line 207: | ||
<li>500 mM NaCl </li> | <li>500 mM NaCl </li> | ||
<li>20 mM imidazole</li> | <li>20 mM imidazole</li> | ||
− | <li>2.5 % glycerol </li> | + | <li>2.5 % (v/v) glycerol </li> |
<li>1 mM DTT</li> | <li>1 mM DTT</li> | ||
</ul> | </ul> | ||
Line 163: | Line 217: | ||
<div class="panel-heading"> | <div class="panel-heading"> | ||
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a data-toggle="collapse" href="# | + | <a data-toggle="collapse" href="#collapseSeven">Elution buffer for purification of LacI</a> |
</h4> | </h4> | ||
</div> | </div> | ||
− | <div id=" | + | <div id="collapseSeven" class="panel-collapse collapse"> |
<div class="panel-body"> | <div class="panel-body"> | ||
<ul> | <ul> | ||
Line 190: | Line 244: | ||
<div class="panel-heading"> | <div class="panel-heading"> | ||
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a data-toggle="collapse" href="# | + | <a data-toggle="collapse" href="#collapseEight">Buffer for dialysis of LacI/ Kpi wash buffer for PRIA</a> |
</h4> | </h4> | ||
</div> | </div> | ||
− | <div id=" | + | <div id="collapseEight" class="panel-collapse collapse"> |
<div class="panel-body"> | <div class="panel-body"> | ||
<ul> | <ul> | ||
Line 206: | Line 260: | ||
<ul> | <ul> | ||
<li>200 mM potassium phosphate</li> | <li>200 mM potassium phosphate</li> | ||
− | <li>5 % glucose </li> | + | <li>5 % (w/v) glucose </li> |
<li>1 mM DTT</li> | <li>1 mM DTT</li> | ||
</ul> | </ul> | ||
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</div> | </div> | ||
</div> | </div> | ||
− | + | <div class="panel panel-default"> | |
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#binding">Binding buffer for PRIA</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="binding" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li>10 mM TRIS-HCl</li> | ||
+ | <li>Adjust pH 8 with hydrogen chloride.</li> | ||
+ | <li>Add 1 mM DTT to the buffer.</li> | ||
+ | <li>Add KCl or NaCl as needed.</li> | ||
+ | <li>For the optimal binding buffer 500 mM KCl is added. </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#hepes">HEPES (storage of proteins)</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="hepes" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li>50 mM HEPES</li> | ||
+ | <li>Adjust pH 7.2 with hydrogen chloride.</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#EMSArunning">EMSA running buffer</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="EMSArunning" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li>20 mM Na<sub>2</sub>HPO<sub>4</sub>, pH 8</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#EMSAreaction">EMSA buffer for reactions</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="EMSAreaction" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li>100 mM sodium phosphate buffer, pH 8.0</li> | ||
+ | <li>375 mM KCl</li> | ||
+ | <li>25% (w/v) glycine</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#columnBuffer">Buffer for CBD-purification</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="columnBuffer" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li>20 mM Na<sub>2</sub>HPO<sub>4</sub>*2 H<sub>2</sub></li> | ||
+ | <li>1.5 M NaCl</li> | ||
+ | <li>1 mM EDTA</li> | ||
+ | <li>Dissolve all substances in water.</li> | ||
+ | <li>Adjust pH 8 with phosphoric acid.</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#abstripping">Antibody stripping buffer</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="abstripping" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li>0.2 M glycine</li> | ||
+ | <li>0.05% (v/v) Tween 20</li> | ||
+ | <li>Adjust pH 2.5 with hydrogen chloride.</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#blockDNA">Blocking solution (DNA immobilization)</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="blockDNA" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li>0.1 M TRIS-HCl, pH 9.0</li> | ||
+ | <li>50 mM ethanolamine</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#milkpowder">Blocking solution (unspecific protein interaction)</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="milkpowder" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li>1x PBS</li> | ||
+ | <li>3% (w/v) milk powder solution</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#annealing">Buffer for annealing of oligonucleotides (PRIA)</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="annealing" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li>50 mM sodium phosphate, pH 8.5</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
<div class="panel-heading"> | <div class="panel-heading"> | ||
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a data-toggle="collapse" href="# | + | <a data-toggle="collapse" href="#collapseNine">Buffers and stock solutions for CFPS</a> |
</h4> | </h4> | ||
</div> | </div> | ||
− | <div id=" | + | <div id="collapseNine" class="panel-collapse collapse"> |
<div class="panel-body"> | <div class="panel-body"> | ||
<h3> Buffers </h3> | <h3> Buffers </h3> | ||
<ul> | <ul> | ||
− | <li> S30A buffer is prepared according to <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Sun2013">Sun et al. 2013</a> and contains | + | <li> S30A buffer is prepared according to <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Sun2013" target="_blank">Sun et al. 2013</a> and contains |
<ul> | <ul> | ||
<li> 14 mM Mg-glutamate</li> | <li> 14 mM Mg-glutamate</li> | ||
Line 237: | Line 440: | ||
<li> 60 mM K-glutamate</li> | <li> 60 mM K-glutamate</li> | ||
<li> 10 mM TRIS</li> | <li> 10 mM TRIS</li> | ||
+ | </ul> | ||
at pH = 8.2 (with acetic acid), autoclaved, stored at 4 °C. Right before use, DTT is added to 2 mM final concentration. </li> | at pH = 8.2 (with acetic acid), autoclaved, stored at 4 °C. Right before use, DTT is added to 2 mM final concentration. </li> | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
− | + | ||
+ | |||
+ | <h3 id="stocksolutions"> Stock solutions </h3> | ||
<ul> | <ul> | ||
− | <li> 1.5 M Spermidine and 1 M putrescine solutions are prepared according to supporting material from <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Yang2012a">Yang et al. 2012a</a>. Briefly, a total of 4.59 mL of autoclaved | + | <li> 1.5 M Spermidine and 1 M putrescine solutions are prepared according to supporting material from <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Yang2012a">Yang et al. 2012a</a>. Briefly, a total of 4.59 mL of autoclaved ddH<sub>2</sub>O is added in small steps to 1 g of spermidine in a bottle. After each step, the bottle is mixed vigorously and the liquid is poured into a beaker where it is continously stirred. After rinsing out the whole bottle, the homogenous solution is aliquoted, flash freezed and stored at -80 °C. The procedure is similar for putrescine, though volumes differ. |
</li> | </li> | ||
− | <li> </li> | + | <li> NTP mixture is prepared from ATP, CTP, GTP and UTP stock solutions (100 mM) from Thermo Scientific. 20x NTP premix contains 30 mM of ATP and GTP, 18 mM CTP and UTP. </li> |
− | + | ||
+ | <li> 330 mM Phosphoenolpyruvate (PEP) stock solution is prepared and pH is adjusted with 5 M KOH according to supporting material from <a href= "https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/CFPS#Yang2012a">Yang et al. 2012a</a> </li> | ||
+ | |||
+ | |||
+ | |||
</ul> | </ul> | ||
+ | |||
Line 257: | Line 468: | ||
</div> | </div> | ||
+ | |||
+ | <h1>Other</h1> | ||
+ | <div class="panel-group" id="accordion"> | ||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#SDSrunning">10x running buffer for SDS-PAGE</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="SDSrunning" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li>250 mM TRIS-HCl</li> | ||
+ | <li>1.92 M Glycine</li> | ||
+ | <li>1 % (w/v) SDS</li> | ||
+ | <li>pH 8.3</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#stainSDS">Staining solution for SDS-PAGE</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="stainSDS" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li>2 g/L Coomassie Brilliant Blue R250</li> | ||
+ | <li>0.5 g/L Coomassie Brilliant Blue G250</li> | ||
+ | <li>25 % (v/v) Isopropanol</li> | ||
+ | <li>10 % (v/v) Acetic acid</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#PBJR">6x PBJR for preparation of SDS-PAGE samples</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="PBJR" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li>7 ml Tris-HCl</li> | ||
+ | <li>3 ml 37 % (v/v) glycerol</li> | ||
+ | <li>0.5 M SDS</li> | ||
+ | <li>0.93 g dithiothreitol</li> | ||
+ | <li>1.2 mg bromophenol blue | ||
+ | |||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#stackingGel">Stacking gel for SDS-PAGE</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="stackingGel" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li>1.3 mL rotiphorese-gel 30</li> | ||
+ | <li>6.1 mL ddH<sub>2</sub>O</li> | ||
+ | <li>2.5 mL 0.5 M TRIS-HCl, pH 6.8</li> | ||
+ | <li>0.1 mL 10% (w/v) SDS</li> | ||
+ | <li> Add 50 µl 10 % (w/v) ammonium persulfate and 5 µl 99 % (v/v) TEMED to each aliquote and mix. </li> | ||
+ | |||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#resolvingGel">Resolving gel for SDS-PAGE</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="resolvingGel" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li>4.7 mL rotiphorese-gel 30</li> | ||
+ | <li>2.7 mL ddH<sub>2</sub>O</li> | ||
+ | <li>2.5 mL 0.5 M TRIS-HCl, pH 6.8</li> | ||
+ | <li>0.1 mL 10 % (w/v) SDS</li> | ||
+ | <li> Add 50 µl 10 % ammonium persulfate (w/v) and 10 µl TEMED and mix. </li> | ||
+ | <li> Pour the solution quickly into the gel casting form. Leave about two centimeters below the bottom of the comb for the stacking gel. </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#collapseTen">SDS destaining solution</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="collapseTen" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li>For 1 liter destaining solution:</li> | ||
+ | <ul> | ||
+ | <li>450 mL ethanol</li> | ||
+ | <li>100 mL acetic acid</li> | ||
+ | <li>Fill the bottle with deionized H<sub>2</sub>O</li><br> | ||
+ | </ul> | ||
+ | |||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#MALDIstandard">MALDI standard solution</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="MALDIstandard" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li>Contains angiotensin II, substance P, adrenocorticotropic hormones, clip 1-17 and clip 18-39</li> | ||
+ | <li>Dissolve 1:25 with water.</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#trypsinsolution">Trypsin solution</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="trypsinsolution" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li> 1 µL trypsin</li> | ||
+ | <li>14 µL 10 mM NH<sub>4</sub>HCO<sub>3</sub></li> | ||
+ | <li>For this solution solubilize lyophilized trypsin in 200 µL of provided buffer and activate trypsin for 15 minutes at 30 °C. For further use it can be stored at -20 °C </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#hccamatrix">HCCA Matrix for MALDI</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="hccamatrix" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li>711 µL 100% acetonitrile</li> | ||
+ | <li>37 µL 0.1% (v/v) TFA</li> | ||
+ | <li>36 µL HCCA stock solution</li> | ||
+ | <li>8 µL 10% (v/v) TFA</li> | ||
+ | <li>8 µL 100 mM NH<sub>4</sub>H<sub>2</sub>PO<sub>4</sub></li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a data-toggle="collapse" href="#paperActivation">PDITC solution for activation of paper</a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="paperActivation" class="panel-collapse collapse"> | ||
+ | <div class="panel-body"> | ||
+ | <ul> | ||
+ | <li><i>p</i>-phenylen-diisothiocyanate</li> | ||
+ | <li>Dissolve in pure ethanol or acetic acid or dried DMSO.</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
+ | </div> | ||
+ | <div class="row footer"> | ||
+ | <div class="col-md-2 col-md-offset-10 text-center"> | ||
+ | <a type="button" class="btn btn-default btn-next" href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Primers"><img src="https://static.igem.org/mediawiki/2015/b/bc/Bielefeld-CeBiTec_notebook_logo.png"><p>Primers</p></a> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | </div> | ||
</body> | </body> | ||
Latest revision as of 21:04, 18 September 2015
Media & Buffers
All of our favorite recipes.
Media
- For 1 liter LB:
- 20 g LB powder (Merck)
- Fill the bottle with deionized H2O
- For 1 L LB-plates:
- 18 g LB powder (Merck)
- 16 g Select Agar (Invitrogen)
- Fill the bottle with deionized H2O
- Add the following components for 900 mL of distilled H2O:
- 20 g Trypton
- 5 g Bacto Yeast Extract
- 2 mL of 5 M NaCl
- 2.5 mL of 1 M KCl
- 10 mL of 1 M MgMgCl2
- 10 mL of 1 M MgSO4
- 20 mL of 1 M glucose
- For 1 L, combine
- 16 g tryptone
- 10 g yeast extract
- 5 g NaCl
- 22 mL KH2PO4
- 40 mL K2HPO4
- dissolve in water and titrate to pH = 7.0 with NaOH, autoclave
Buffers
- For each litre of solution:
- 242 g TRIS Base (MW=121.1)
- 57.1 ml Glacial Acetic Acid
- 100 ml 0.5 M EDTA
- mix Tris with stir bar to dissolve in about 600 ml of ddH2O.
- add the EDTA and Acetic Acid.
- bring final volume to 1 l with ddH20.
- store at room temperature.
- Note: Final (1x) working concentration:
- 0.04 M Tris - Acetate
- 0.001 M EDTA
- For each 200 ml of solution:
- 5.844 g NaCl
- 0.272 g imidazole
- 5.8 ml of 86 % (v/v) glycerol
- 0.406 g MgCl2
- 0.2 g lysozyme
- Add all these ingredients to a 50 mM sodium phosphate buffer (pH 8.0). Prior to use add DTT to a final concentration of 1 mM and tween 20 to a final concentration of 0.1 % (v/v).
- store at room temperature.
- Note: Final (1x) working concentration:
- 50 mM Sodiumphosphate
- 500 mM NaCl
- 20 mM imidazole
- 2.5 % glycerol
- 1 mM DTT
- 10 mM MgCl2
- 0.1 % (v/v) tween 20
- 1 mg/mL lysozyme
- For each litre of solution:
- 29.22 g NaCl
- 1.36 g imidazole
- 29 ml of 86 % (v/v) glycerol
- Add all these ingredients to a 50 mM sodium phosphate buffer (pH 8.0). Prior to use add DTT to a final concentration of 1 mM.
- store at room temperature.
- Note: Final (1x) working concentration:
- 50 mM sodium phosphate
- 500 mM NaCl
- 20 mM imidazole
- 2.5 % (v/v) glycerol
- 1 mM DTT
- For each litre of solution:
- 17.53 g NaCl
- 13.6 g imidazole
- Add all these ingredients to a 20 mM sodium phosphate buffer (pH 7.4).
- store at room temperature.
- Note: Final (1x) working concentration:
- 20 mM sodium phosphate
- 300 mM NaCl
- 200 mM imidazole
- For each litre of solution:
- 50 g glucose
- Add the glucose to a 200 mM potassium phosphate buffer (pH 7.6). Prior to usage add DTT to a final concentration of 1 mM
- store at room temperature.
- Note: Final (1x) working concentration:
- 200 mM potassium phosphate
- 5 % (w/v) glucose
- 1 mM DTT
- 10 mM TRIS-HCl
- Adjust pH 8 with hydrogen chloride.
- Add 1 mM DTT to the buffer.
- Add KCl or NaCl as needed.
- For the optimal binding buffer 500 mM KCl is added.
- 50 mM HEPES
- Adjust pH 7.2 with hydrogen chloride.
- 20 mM Na2HPO4, pH 8
- 100 mM sodium phosphate buffer, pH 8.0
- 375 mM KCl
- 25% (w/v) glycine
- 20 mM Na2HPO4*2 H2
- 1.5 M NaCl
- 1 mM EDTA
- Dissolve all substances in water.
- Adjust pH 8 with phosphoric acid.
- 0.2 M glycine
- 0.05% (v/v) Tween 20
- Adjust pH 2.5 with hydrogen chloride.
- 0.1 M TRIS-HCl, pH 9.0
- 50 mM ethanolamine
- 1x PBS
- 3% (w/v) milk powder solution
- 50 mM sodium phosphate, pH 8.5
Buffers
- S30A buffer is prepared according to Sun et al. 2013 and contains
- 14 mM Mg-glutamate
- 60 mM K-glutamate
- 50 mM TRIS
- S30 buffer (buffer A from Kwon and Jewett 2015) with lower TRIS concentration
- 14 mM Mg-glutamate
- 60 mM K-glutamate
- 10 mM TRIS
Stock solutions
- 1.5 M Spermidine and 1 M putrescine solutions are prepared according to supporting material from Yang et al. 2012a. Briefly, a total of 4.59 mL of autoclaved ddH2O is added in small steps to 1 g of spermidine in a bottle. After each step, the bottle is mixed vigorously and the liquid is poured into a beaker where it is continously stirred. After rinsing out the whole bottle, the homogenous solution is aliquoted, flash freezed and stored at -80 °C. The procedure is similar for putrescine, though volumes differ.
- NTP mixture is prepared from ATP, CTP, GTP and UTP stock solutions (100 mM) from Thermo Scientific. 20x NTP premix contains 30 mM of ATP and GTP, 18 mM CTP and UTP.
- 330 mM Phosphoenolpyruvate (PEP) stock solution is prepared and pH is adjusted with 5 M KOH according to supporting material from Yang et al. 2012a
Other
- 250 mM TRIS-HCl
- 1.92 M Glycine
- 1 % (w/v) SDS
- pH 8.3
- 2 g/L Coomassie Brilliant Blue R250
- 0.5 g/L Coomassie Brilliant Blue G250
- 25 % (v/v) Isopropanol
- 10 % (v/v) Acetic acid
- 7 ml Tris-HCl
- 3 ml 37 % (v/v) glycerol
- 0.5 M SDS
- 0.93 g dithiothreitol
- 1.2 mg bromophenol blue
- 1.3 mL rotiphorese-gel 30
- 6.1 mL ddH2O
- 2.5 mL 0.5 M TRIS-HCl, pH 6.8
- 0.1 mL 10% (w/v) SDS
- Add 50 µl 10 % (w/v) ammonium persulfate and 5 µl 99 % (v/v) TEMED to each aliquote and mix.
- 4.7 mL rotiphorese-gel 30
- 2.7 mL ddH2O
- 2.5 mL 0.5 M TRIS-HCl, pH 6.8
- 0.1 mL 10 % (w/v) SDS
- Add 50 µl 10 % ammonium persulfate (w/v) and 10 µl TEMED and mix.
- Pour the solution quickly into the gel casting form. Leave about two centimeters below the bottom of the comb for the stacking gel.
- For 1 liter destaining solution:
- 450 mL ethanol
- 100 mL acetic acid
- Fill the bottle with deionized H2O
- Contains angiotensin II, substance P, adrenocorticotropic hormones, clip 1-17 and clip 18-39
- Dissolve 1:25 with water.
- 1 µL trypsin
- 14 µL 10 mM NH4HCO3
- For this solution solubilize lyophilized trypsin in 200 µL of provided buffer and activate trypsin for 15 minutes at 30 °C. For further use it can be stored at -20 °C
- 711 µL 100% acetonitrile
- 37 µL 0.1% (v/v) TFA
- 36 µL HCCA stock solution
- 8 µL 10% (v/v) TFA
- 8 µL 100 mM NH4H2PO4
- p-phenylen-diisothiocyanate
- Dissolve in pure ethanol or acetic acid or dried DMSO.