Team:UIUC-Illinois/Notebook/Protocols

From 2012.igem.org

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<h1><center>Select Protocol</center></h1>
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/Bootcamp">Bootcamp Protocols</a>
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/Subculturing">Subculturing Plates</a>
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/Electrocompetence">Making Electrocompetent E.Coli</a>
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/TAE">Making TAE Buffers</a>
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                    <li><a name="prot1" >Bootcamp Protocols</a></li>
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                    <li><a name="prot2" >Digestions</a></li>
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/GelElectrophoresis">Making Electrophoresis Gels</a>
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                    <li><a name="prot3" >Gel Purification</a></li>
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                    <li><a name="prot4" >Inoculation</a></li>
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                    <li><a name="prot5" >Ligation</a></li>
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                    <li><a name="prot6" >Making Electrocompetent E.Coli</a></li>
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/GelPurification">Gel Purification</a>
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                    <li><a name="prot7" >Making Electrophoresis Gels</a></li>
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                    <li><a name="prot8" >Making TAE Buffers</a></li>
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                    <li><a name="prot9" >Miniprep</a></li>
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                    <li><a name="prot10" >PCR Protocols</a></li>
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/Inoculation">Inoculation</a>
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                    <li><a name="prot11" >Storage of Cells</a></li>
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                    <li><a name="prot12" >Subculturing Plates</a></li>
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                    <li><a name="prot13" >Transformation of E.Coli</a></li>
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/Miniprep">Miniprep</a>
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/PCR">PCR Protocols</a>
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/Digestions">Digestions</a>
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3
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<div id="prot4" style="display:none">
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/Ligations">Ligations</a>
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/Transformation">Transformation of E.Coli</a>
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/Storage">Storage of Cells</a>
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<div id="prot8" style="display:none">
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<div id="prot10" style="display:none">
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<div id="prot11" style="display:none">
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<center><h1>Bootcamp Protocols</h1></center>
 
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<h2>Making LB for plates</h2> <br/>
 
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To make 1 Liter of LB: <br/><br/>
 
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1. Always use a 10:5:5 ratio of Tryptone to Yeast Extract to NaCl. <br/>
 
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2. Add dry ingredients first. <br/>
 
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3. Use a 2L flask. <br/> <br/>
 
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4. Add the following <br/><br/>
 
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- 10 g Tryptone <br/>
 
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- 5 g Yeast Extract <br/>
 
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- 5 g NaCl <br/>
 
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- 1.5 g Agar (NOT agarose!)<br/><br/>
 
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4. Then add 1 L of MilliQ water. <br/>
 
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5. Autoclave by total volume.<br/>
 
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6. Pour 25 mL on each plate (just enough to cover the bottom). <br/><br/>
 
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<h2>Making Liquid Media </h2> <br/>
 
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1. Always use a 5-2.5-2.5 ratio of Tryptone to Yeast Extract to NaCl <br/>
 
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2. Add dry ingredients first, then add MilliQ water <br/>
 
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3. No Agar! <br/>
 
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4. Autoclave by total volume <br/><br/>
 
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<h2>Making Glycerol Stock </h2> <br/>
 
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To make 400 mL of 10% glycerol you will need:<br/><br/>
 
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-40 mL glycerol<br/>
 
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-360 mL of MilliQ water <br/><br/>
 
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To make 400 mL of 20% glycerol you will need:<br/><br/>
 
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-80 mL glycerol<br/>
 
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-320 mL of MilliQ water <br/><br/>
 
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1. Use flasks and bottles before graduated cylinders (they take forever to mix!)<br/>
 
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2. Need a stir plate and a large stir bar <br/>
 
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3. Stir until mixture is homogeneous <br/>
 
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4. Don’t autoclave! <br/><br/>
 
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<h2>Making ddH2O</h2> <br/>
 
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- Use the small bottles. <br/>
 
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- Autoclave water by total volume. <br/><br/>
 
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<h2>Autoclaving </h2><br/>
 
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- Robert is the source of official help on all things about the Autoclave. <br/><br/>
 
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1. Use the autoclave tape. <br/>
 
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2. Look at the Betastar for settings. <br/>
 
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3. Go by materials and total volume. <br/>
 
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4. Make sure the autoclave tape has changed colour by the end of the cycle. Check conditions and settings then repeat steps 1-4 if tape has not changed colour. <br/><br/>
 
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<h2>Ligation </h2><br/>
 
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We used the <a href="http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf">Ginkgo bioworks protocol</a> <br/><br/>
 
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1. Control: <br/><br/>
 
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- 2 uL of Circular PSB1C3 plasmid 13 uL ddH2O <br/>
 
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- 2 uL T4 ligase buffer<br/>
 
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- 1 uL T4 ligase <br/><br/>
 
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2. P&C: <br/><br/>
 
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- 2 uL of Circular PSB1C3 plasmid<br/>
 
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- 2 uL of PUF, 11 uL ddH2O<br/>
 
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- 2 uL T4 ligase buffer<br/>
 
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- 1 uL T4 ligase<br/><br/>
 
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3. L&P:<br/><br/>
 
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- 2 uL of linear PSB1C3 plasmid<br/>
 
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- 2 uL of PUF<br/>
 
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- 11 uL ddH2O<br/>
 
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- 2 uL T4 ligase buffer<br/>
 
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- 1 uL T4 ligase<br/><br/>
 
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Revision as of 19:28, 7 June 2012

Header

Protocols

  • Bootcamp Protocols
  • Digestions
  • Gel Purification
  • Inoculation
  • Ligation
  • Making Electrocompetent E.Coli
  • Making Electrophoresis Gels
  • Making TAE Buffers
  • Miniprep
  • PCR Protocols
  • Storage of Cells
  • Subculturing Plates
  • Transformation of E.Coli

    • Retrieved from "http://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols"