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| − | <title>Kanamycin and cortisol agar plate prep · Benchling</title> | + | <title>LB Broth and Agar Plates · Benchling</title> |
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| − | <body><div><svg id="__SVG_SPRITE_NODE__" style="position: absolute; width: 0; height: 0" xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink"></svg><div><div data-reactroot=""><div class="printable-container hide-print-links"><div><div class="protocolEditor"><div><!-- react-empty: 6 --><!-- react-empty: 7 --></div><div class="protocolEditor-pageContainer"><div class="protocolEditor-page"><div class="protocolEditor-contentWrapper"><h1 class="mediocre-titleEditor is-locked"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="pv955-title" data-index="0">Kanamycin and cortisol agar plate prep</div></h1><div><h2>Introduction</h2><div class="intro-editor" data-editor="intro"><div class="editable selectable is-locked" contenteditable="false" data-allow-multiline="true" data-cid="pv955-intro" data-index="0">Addgene.
| + | <body><div><svg id="__SVG_SPRITE_NODE__" style="position: absolute; width: 0; height: 0" xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink"></svg><div><div data-reactroot=""><div class="printable-container hide-print-links"><div><div class="printable-page-container"><div class="noteEditor mediocre" tabindex="-1"><div><!-- react-empty: 7 --><!-- react-empty: 8 --><!-- react-empty: 9 --><!-- react-empty: 10 --><!-- react-empty: 11 --></div><div class="noteEditor-container"><div class="u-cursor-text noteEditor-catcher u-disable-margin-collapse dnd-drop-container"><div class="noteEditor-contentWrapper" data-mediocre-item-whitespace="1"><h1 class="mediocre-titleEditor"><span class="mediocre-titleEditor-header"><span class="mediocre-titleEditor-header-id u-faded"></span></span><div class="mediocre-titleEditor-titleEditable editable selectable is-locked" contenteditable="false" data-allow-multiline="false" data-cid="notetitleeditor" data-index="0">LB Broth and Agar Plates</div></h1><div><div><b>Project:</b><!-- react-text: 22 --> <!-- /react-text --><!-- react-text: 23 -->QGEM Team<!-- /react-text --></div><div><b>Authors:</b><!-- react-text: 26 --> <!-- /react-text --><!-- react-text: 27 -->Ellis Kelly<!-- /react-text --></div><div><b>Date:</b><!-- react-text: 30 --> <!-- /react-text --><!-- react-text: 31 -->2018-05-22<!-- /react-text --></div></div><!-- react-empty: 32 --><div class="editor" data-editor="notes"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="daySeparator" data-scroll-id="entryDay980"><div class="daySeparator-inputWrapper"><input class="daySeparator-inputWrapper-hiddenInput" type="input"/></div><span class="daySeparator-date">Tuesday, 5/22/18</span><div class="daySeparator-line"></div></div><div class="mediocre-item is-text" data-scroll-id="note-part984"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 0px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part984" data-index="0" style="margin-left: 0px;"><b>LB Broth (1 L) - 2 bottles of 500mL each, with Don's Media</b></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part985"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part985" data-index="1" style="margin-left: 35px;"><u>Materials: </u></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part986"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part986" data-index="2" style="margin-left: 70px;">10 g Bacto-Tryptone</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part987"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part987" data-index="3" style="margin-left: 70px;">5 g Bacto-Yeast Extract</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part988"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part988" data-index="4" style="margin-left: 70px;">10 g NaCl </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part989"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part989" data-index="5" style="margin-left: 70px;">1 L ddH2O</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part990"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part990" data-index="6" style="margin-left: 35px;"><u>Procedure:</u></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part991"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part991" data-index="7" style="margin-left: 70px;">Add 900mL of ddH2O to a large beaker with a stir bar. Add Tryptone, Yeast Extract, NaCl. Measure the pH with indicator strips. If the pH is 7.0, add another 100 mL of ddH2O. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part992"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 105px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part992" data-index="8" style="margin-left: 105px;">** When using Tryptone, wear a face mask and goggles. It is dry and very powdery and dangerous to inhale. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part993"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part993" data-index="9" style="margin-left: 70px;">Add the mixture to 2X 500mL glass bottles. Add autoclave tape and autoclave for 30 min. <span style="color:#D10E0E">******MAKE SURE TO LOOSEN THE LIDS BEFORE AUTOCLAVING</span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part994"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 0px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part994" data-index="10" style="margin-left: 0px;"><b>LB Agar Broth (1L): </b></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part995"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part995" data-index="11" style="margin-left: 35px;"><u>Materials:</u></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part996"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part996" data-index="12" style="margin-left: 70px;">10 g Bacto-Tryptone</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part997"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part997" data-index="13" style="margin-left: 70px;">5 g Bacto-Yeast Extract</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part998"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part998" data-index="14" style="margin-left: 70px;">10 g NaCl </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part999"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part999" data-index="15" style="margin-left: 70px;">ddH2O</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1000"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1000" data-index="16" style="margin-left: 70px;">15 g of Bacto-agar</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1001"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1001" data-index="17" style="margin-left: 35px;"><u>Procedure:</u></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1002"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1002" data-index="18" style="margin-left: 70px;"><span style="color:#344043"><span style="background-color:#FFFFFF">Add 900mL of ddH2O to a large beaker with a stir bar. Add Tryptone, Yeast Extract, NaCl and agar. Measure the pH with indicator strips. If the pH is 7.0, add ddH2O until the total volume is 1L. </span></span></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1003"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1003" data-index="19" style="margin-left: 70px;">Add to a 1L glass bottle, pour in a little at a time and then swirl to ensure agar stays in solution. Autoclave. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1004"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 0px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1004" data-index="20" style="margin-left: 0px;"><b>Preparing Ampicillin:</b></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1005"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1005" data-index="21" style="margin-left: 35px;"><u>Materials: </u></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1006"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1006" data-index="22" style="margin-left: 70px;">1 g of Ampicillin</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1007"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1007" data-index="23" style="margin-left: 70px;">ddH20</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1008"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1008" data-index="24" style="margin-left: 70px;">15mL tube</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1009"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1009" data-index="25" style="margin-left: 70px;">autoclaved tubes </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1010"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1010" data-index="26" style="margin-left: 35px;"><u>Procedure: </u></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1011"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1011" data-index="27" style="margin-left: 70px;">Weigh out ampicillin on weigh sheet. Add to 15mL tube. Add ddH2O until volume reaches 10 mL. Add a few mL's at a time and then swirl to ensure the ampicillin dissolves. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1012"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1012" data-index="28" style="margin-left: 70px;">Aliquot final liquid into 500uL tubes. Store at -20C. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1013"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 0px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1013" data-index="29" style="margin-left: 0px;"><b>Preparing Ampicillin Plates:</b></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1014"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1014" data-index="30" style="margin-left: 35px;"><u>Materials:</u></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1015"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1015" data-index="31" style="margin-left: 70px;">LB Agar Broth</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1016"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1016" data-index="32" style="margin-left: 70px;">Ampicillin</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1017"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1017" data-index="33" style="margin-left: 70px;">Plastic plates</div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1018"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 35px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1018" data-index="34" style="margin-left: 35px;"><u>Procedure: </u></div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1019"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1019" data-index="35" style="margin-left: 70px;">Let the LB agar broth cool until it is warm, but safe to touch (too hot and the ampicillin will be ineffective, too cool and you will have difficulty pouring the plates). </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1020"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1020" data-index="36" style="margin-left: 70px;">Obtain 1000uL of ampicillin and let thaw. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1021"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1021" data-index="37" style="margin-left: 70px;">Start a flame. Keep all the materials within close proximity to the flame. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1022"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1022" data-index="38" style="margin-left: 70px;">Add the ampicillin to the LB agar broth. Shake. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1023"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1023" data-index="39" style="margin-left: 70px;">Slowly pour the agar into the plates until the bottom is just covered. Flame any bubbles - not too long otherwise the ampicillin will be ineffective. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1024"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1024" data-index="40" style="margin-left: 70px;">Let the plates cool for a few minutes with the lid half covering the plate. This prevents too much condensation from occuring. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1025"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1025" data-index="41" style="margin-left: 70px;">After a few minutes, put the lid on fully. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1026"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 70px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1026" data-index="42" style="margin-left: 70px;">Let the plates set overnight and store at -4C. </div></div></div><div class="mediocre-item is-text" data-scroll-id="note-part1027"><div class="mediocre-noteEditable multi-editable is-paragraph"><div class="mediocre-noteEditable-indenter" style="width: 0px;"></div><div class="editable selectable is-locked" contenteditable="false" data-allow-at-search="true" data-allow-multiline="false" data-cid="note-part1027" data-index="43" style="margin-left: 0px;"></div></div></div></div></div><div class="dnd-drop-container-border"></div></div></div><div class="noteEditor-nueModuleContainer"><!-- react-empty: 218 --></div></div></div></div></div><!-- react-empty: 219 --></div></div></div> |
| − | </div></div><h2>Materials</h2><div class="materials-editor" data-editor="materials"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><ul class="materials"><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m956" data-index="0">37g pre-mixed powder consisting of:</div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c957" data-index="0,0">5 g peptone</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c958" data-index="0,1">10.0 g peptone from casein</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c959" data-index="0,2">10.0 g sodium chloride</div></li><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c960" data-index="0,3">12.0 g agar-agar</div></li></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m961" data-index="1">1.0 L Sterile H2O</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m962" data-index="2">Sterile plates of your desired size (usually 60 mm x 15 mm plates) which can hold 5-10 mL of agar on which you can individually distinguish a maximum of ~200 colonies. </div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m963" data-index="3">Autoclave flasks</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m964" data-index="4">Sterile pipettes</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m965" data-index="5">Ice bucket to hold antibiotics</div><ul class="components"></ul></li><li class="material"><div class="material-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="m966" data-index="6">Antibiotic at 1000 x concentration dissolved in the appropriate liquid solvent. </div><ul class="components"><li class="component"><div class="component-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="c967" data-index="6,0">Kanamycin --> stock concentrationL 50 mg/mL, recommened working concentration (50µg/mL). </div></li></ul></li></ul></div><h2>Procedure</h2><div class="stepsEditor mediocre"><div class="editor" data-editor="steps"><div class="hiddenFocusEditable js-focusBearer" contenteditable="true" tabindex="-1"></div><div class="mediocre-item is-header" data-scroll-id="s968"><ul class="headers"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s968" data-index="0"></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s968" data-index="0" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s969"><ol class="steps"><li value="1"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s969" data-index="1">Measure 37 g of pre-mixed LB-agar powder per L molten agar you'd like to make. The precise mass you measure out will be based on the number of plates you'd like to pour. </div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s969" data-index="1" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s970"><ol class="steps"><li value="2"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s970" data-index="2">Transfer the LB-agar powder you've measured out into an appropriately sized bottle for autoclaving. We make 400 mL of agar <span style="color:#333333"><span style="background-color:#FFFFFF">in 1 L bottles and 200 mL of agar in 500 mL bottles. The extra empty volume is necessary to prevent your molten agar from boiling over in the autoclave</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s970" data-index="2" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s971"><ol class="steps"><li value="3"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s971" data-index="3"><span style="color:#333333"><span style="background-color:#FFFFFF">Transfer the sterile water to the same bottle and swirl to form a medium/agar colloid</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s971" data-index="3" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s972"><ol class="steps"><li value="4"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s972" data-index="4">Cover the opening of the bottle with its cap or aluminum foil (but do not make an air-tight seal!) and tape the bottle with autoclave tape. The autoclave tape will darken during the autoclave process if your sample has spent at least 10 min at 121 ℃. Use lab tape to label the bottle with your initials, the date, and the bottle contents. This will clear up any confusion later if your forget your bottle in the autoclave.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s972" data-index="4" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s973"><ol class="steps"><li value="5"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s973" data-index="5"><span style="color:#333333"><span style="background-color:#FFFFFF">Place the gel mix in the autoclave and run on a setting that gets the sample to at least 121 ℃ under 20 psi for at least 30 min. The high pressure will prevent your gel mix from boiling over at high temperature.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s973" data-index="5" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s974"><ol class="steps"><li value="6"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s974" data-index="6"><span style="color:#333333"><span style="background-color:#FFFFFF">While your samples are sterilizing in the autoclave, you should prepare your plate pouring station:</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s974" data-index="6" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description" data-scroll-id="s975"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s975" data-index="7">- Find an empty section of lab bench with a working flame.</div></div></li></ul></div><div class="mediocre-item is-description" data-scroll-id="s976"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s976" data-index="8">- Spray down the bench with a 70% ethanol solution and wipe down with a paper towel.</div></div></li></ul></div><div class="mediocre-item is-description" data-scroll-id="s977"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s977" data-index="9">- Count out the appropriate number of plates and stack them on your lab bench.</div></div></li></ul></div><div class="mediocre-item is-description" data-scroll-id="s978"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s978" data-index="10">- <span style="color:#333333"><span style="background-color:#FFFFFF">Label the plates with the date and the medium they will contain including the identity of the antibiotic</span></span></div></div></li></ul></div><div class="mediocre-item is-description" data-scroll-id="s979"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s979" data-index="11">- Position the flame just to the side of where you’ll be pouring your plates - be sure to leave room for your bottle of molten gel mix, a tube rack containing the appropriate antibiotics, and a section for active pouring.</div></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s980"><ol class="steps"><li value="7"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s980" data-index="12">Prepare your antibiotics. Prior to adding your antibiotic to the molten gel mix, you should create a 1000x stock solution.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s980" data-index="12" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description" data-scroll-id="s981"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s981" data-index="13">- <b><span style="color:#333333"><span style="background-color:#FFFFFF">For example:</span></span></b><span style="color:#333333"><span style="background-color:#FFFFFF"> If you’ll be preparing plates with a final concentration of 100 ug/mL ampicillin, you should make a stock solution of 100,000 ug/mL (100 mg/mL). Simply measure out 100 mg of ampicillin powder, add it to 1 mL of water, dissolve by vortexing, and filter sterilize.</span></span></div></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s982"><ol class="steps"><li value="8"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s982" data-index="14"><span style="color:#333333"><span style="background-color:#FFFFFF">Prepare a water bath at 60 ℃ with sufficient water to submerge ~75% of the bottle containing your molten gel mixture.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s982" data-index="14" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description" data-scroll-id="s983"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s983" data-index="15">- Make sure that kanamycin doesn't breakdown at this temperature. </div></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s984"><ol class="steps"><li value="9"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s984" data-index="16"><span style="color:#333333"><span style="background-color:#FFFFFF">Retrieve your molten agar mix from the autoclave.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s984" data-index="16" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s985"><ol class="steps"><li value="10"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s985" data-index="17">Partially submerge your molten gel-mix in the 60 ℃ water bath.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s985" data-index="17" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description" data-scroll-id="s986"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s986" data-index="18">- <span style="color:#333333"><span style="background-color:#FFFFFF">You should leave the molten gel-mix in the water bath for at least 5 min. Do not let any of the water bath water touch the neck or top of the bottle as this water is not likely to be sterile. Cooled agar should be warm to the touch; as a rule of thumb, if you cannot take the molten agar out of the water bath wearing only lab gloves, it’s not likely cool enough to add antibiotic to. To be certain your agar is at the right temperature, we recommend using a laser thermometer.</span></span></div></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s987"><ol class="steps"><li value="11"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s987" data-index="19">Light the flame at the plate pouring station and dilute your antibiotic into your ~60 ℃ molten gel mix using sterile technique.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s987" data-index="19" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s988"><ol class="steps"><li value="12"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s988" data-index="20">Swirl the agar bottle to ensure even distribution of the antibiotic throughout the agar.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s988" data-index="20" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s989"><ol class="steps"><li value="13"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s989" data-index="21">Open one plate at a time next to the flame and begin pouring. Measure your desired amount of agar with a pipette for the first plate to get a good idea of what that volume looks like in your particular plate.</div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s989" data-index="21" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-step" data-scroll-id="s990"><ol class="steps"><li value="14"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s990" data-index="22"><span style="color:#333333"><span style="background-color:#FFFFFF">For the remainder of the plates, pour directly from the bottle.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s990" data-index="22" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description" data-scroll-id="s991"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s991" data-index="23">- <span style="color:#333333"><span style="background-color:#FFFFFF">Be sure to swirl your plates after pouring to remove bubbles and ensure an even distrubtion of agar over the bottom of the plate. Cap each plate after pouring and stack as you pour</span></span></div></div></li></ul></div><div class="mediocre-item is-description" data-scroll-id="s992"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s992" data-index="24">- <span style="color:#333333"><span style="background-color:#FFFFFF">If your agar partially solidifies in the bottle while you’re pouring, you should stop pouring and re-make the gel-mix. If you’re making plates without any antibiotic you can alternatively re-liquefy the agar by running it through the autoclave again or by microwaving (if you microwave, beware of over-boiling!).</span></span></div></div></li></ul></div><div class="mediocre-item is-description" data-scroll-id="s993"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s993" data-index="25"></div></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s994"><ol class="steps"><li value="15"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s994" data-index="26"><span style="color:#333333"><span style="background-color:#FFFFFF">Leave your plates out on the bench to solidify.</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s994" data-index="26" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description" data-scroll-id="s995"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s995" data-index="27"><span style="color:#333333"><span style="background-color:#FFFFFF">- It takes roughly 30 min for our plates to solidify at room temperature, however we leave them out at room temperature overnight to allow them to dry. After overnight drying, we place the plates in a plastic bag with an absorbent material to reduce condensation. The plates are then stored at 4 ℃ until use.</span></span></div></div></li></ul></div><div class="mediocre-item is-step" data-scroll-id="s996"><ol class="steps"><li value="16"><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s996" data-index="28"><span style="color:#333333"><span style="background-color:#FFFFFF">Once your plates have solidified and dried, you should test them to make sure the antibiotic functions properly</span></span></div></div><div class="time-editable-wrapper"><div class="time-editable force-visible editable selectable is-locked" contenteditable="false" data-cid="time-s996" data-index="28" data-original-title="" title=""></div><span class="glyphicon glyphicon-time time-editable-wrapper-icon" icon="time"></span></div></li></ol></div><div class="mediocre-item is-description" data-scroll-id="s997"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s997" data-index="29">- T<span style="color:#333333"><span style="background-color:#FFFFFF">ake out two plates.</span></span></div></div></li></ul></div><div class="mediocre-item is-description" data-scroll-id="s998"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s998" data-index="30">- On the first plate, streak out a strain that you know to be resistant to the antibiotic.</div></div></li></ul></div><div class="mediocre-item is-description" data-scroll-id="s999"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s999" data-index="31">- On the second plate, streak out a strain that’s not resistant to the antibiotic.</div></div></li></ul></div><div class="mediocre-item is-description" data-scroll-id="s1000"><ul class="descriptions"><li><div class="step-editable-wrapper"><div class="step-editable multi-editable editable selectable is-locked" contenteditable="false" data-cid="s1000" data-index="32">- <span style="color:#333333"><span style="background-color:#FFFFFF">Incubate both plates overnight at the appropriate growth temperature and check for growth. See our sample data section below for positive and negative test results</span></span></div></div></li></ul></div></div></div></div></div></div></div></div></div></div><!-- react-empty: 272 --></div></div></div>
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