Team:UIUC-Illinois/Notebook/Protocols

From 2012.igem.org

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<div id="protocol-container">
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<div id="protocolselection">
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<h1><center>Select Protocol</center></h1>
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<ul class="protocolclass1">
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<li>
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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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    <div id="protocol-container">
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<li>
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            <div id="protocolmenu">
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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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                    <li><a name="prot1" >Bootcamp Protocols</a></li>
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</li>
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                    <li><a name="prot2" >Digestions</a></li>
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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" >Ligations</a></li>
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                    <li><a name="prot6" >Making Electrocompetent E.Coli</a></li>
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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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                    <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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            </div>
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<div id="protocoldescription">
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<li>
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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>
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<div id="prot1" style="display:none">
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<li>
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Content1
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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>
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<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>
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<li>
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/Inoculation">Inoculation</a>
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</li>
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<li>
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/Miniprep">Miniprep</a>
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</li>
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<li>
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/PCR">PCR Protocols</a>
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</li>
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<li>
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/Digestions">Digestions</a>
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</li>
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<li>
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/Ligations">Ligations</a>
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</li>
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<li>
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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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</li>
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<li>
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<a href="https://2012.igem.org/Team:UIUC-Illinois/Notebook/Protocols/Storage">Storage of Cells</a>
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</li>
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</ul>
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</div>
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<div id="prot2" style="display:none">
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Content1
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<div id="protocoloverview">
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<center><h1>Bootcamp Protocols</h1></center>
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<div id="prot3" style="display:none">
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Content1
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<h2>Making LB for plates</h2> <br/>
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</div>
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To make 1 Liter of LB: <br/><br/>
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<div id="prot4" style="display:none">
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1. Always use a 10:5:5 ratio of Tryptone to Yeast Extract to NaCl. <br/>
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Content1
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2. Add dry ingredients first. <br/>
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</div>
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3. Use a 2L flask. <br/> <br/>
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<div id="prot5" style="display:none">
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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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<div id="prot6" style="display:none">
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- 5 g NaCl <br/>
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Content1
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- 1.5 g Agar (NOT agarose!)<br/><br/>
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</div>
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4. Then add 1 L of MilliQ water. <br/>
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<div id="prot7" style="display:none">
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5. Autoclave by total volume.<br/>
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Content1
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6. Pour 25 mL on each plate (just enough to cover the bottom). <br/><br/>
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</div>
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<div id="prot8" style="display:none">
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<h2>Making Liquid Media </h2> <br/>
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Content1
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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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</div>
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2. Add dry ingredients first, then add MilliQ water <br/>
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<div id="prot9" style="display:none">
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3. No Agar! <br/>
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Content1
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4. Autoclave by total volume <br/><br/>
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</div>
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<div id="prot10" style="display:none">
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<h2>Making Glycerol Stock </h2> <br/>
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Content1
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To make 400 mL of 10% glycerol you will need:<br/><br/>
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</div>
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<div id="prot11" style="display:none">
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-40 mL glycerol<br/>
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Content1
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-360 mL of MilliQ water <br/><br/>
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</div>
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<div id="prot12" style="display:none">
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To make 400 mL of 20% glycerol you will need:<br/><br/>
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Content1
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</div>
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-80 mL glycerol<br/>  
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<div id="prot13" style="display:none">
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-320 mL of MilliQ water <br/><br/>
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Content1
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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/>
 +
 
 +
 
</div>
</div>
</div>
</div>
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</body>

Revision as of 23:26, 6 June 2012

Header

Protocols

Select Protocol

Bootcamp Protocols

Making LB for plates


To make 1 Liter of LB:

1. Always use a 10:5:5 ratio of Tryptone to Yeast Extract to NaCl.
2. Add dry ingredients first.
3. Use a 2L flask.

4. Add the following

- 10 g Tryptone
- 5 g Yeast Extract
- 5 g NaCl
- 1.5 g Agar (NOT agarose!)

4. Then add 1 L of MilliQ water.
5. Autoclave by total volume.
6. Pour 25 mL on each plate (just enough to cover the bottom).

Making Liquid Media


1. Always use a 5-2.5-2.5 ratio of Tryptone to Yeast Extract to NaCl
2. Add dry ingredients first, then add MilliQ water
3. No Agar!
4. Autoclave by total volume

Making Glycerol Stock


To make 400 mL of 10% glycerol you will need:

-40 mL glycerol
-360 mL of MilliQ water

To make 400 mL of 20% glycerol you will need:

-80 mL glycerol
-320 mL of MilliQ water

1. Use flasks and bottles before graduated cylinders (they take forever to mix!)
2. Need a stir plate and a large stir bar
3. Stir until mixture is homogeneous
4. Don’t autoclave!

Making ddH2O


- Use the small bottles.
- Autoclave water by total volume.

Autoclaving


- Robert is the source of official help on all things about the Autoclave.

1. Use the autoclave tape.
2. Look at the Betastar for settings.
3. Go by materials and total volume.
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.

Ligation


We used the Ginkgo bioworks protocol

1. Control:

- 2 uL of Circular PSB1C3 plasmid 13 uL ddH2O
- 2 uL T4 ligase buffer
- 1 uL T4 ligase

2. P&C:

- 2 uL of Circular PSB1C3 plasmid
- 2 uL of PUF, 11 uL ddH2O
- 2 uL T4 ligase buffer
- 1 uL T4 ligase

3. L&P:

- 2 uL of linear PSB1C3 plasmid
- 2 uL of PUF
- 11 uL ddH2O
- 2 uL T4 ligase buffer
- 1 uL T4 ligase

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