Team:UIUC-Illinois/Notebook/Protocols

From 2012.igem.org

(Difference between revisions)
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             <div id="protocoloverview">
             <div id="protocoloverview">
 +
<center><h1>Bootcamp Protocols</h1></center>
 +
<h2>Making LB for plates</h2> <br/>
 +
To make 1 Liter of LB: <br/><br/>
 +
1. Always use a 10:5:5 ratio of Tryptone to Yeast Extract to NaCl. <br/>
 +
2. Add dry ingredients first. <br/>
 +
3. Use a 2L flask. <br/> <br/>
 +
4. Add the following <br/><br/>
 +
- 10 g Tryptone <br/>
 +
- 5 g Yeast Extract <br/>
 +
- 5 g NaCl <br/>
 +
- 1.5 g Agar (NOT agarose!)<br/><br/>
 +
4. Then add 1 L of MilliQ water. <br/>
 +
5. Autoclave by total volume.<br/>
 +
6. Pour 25 mL on each plate (just enough to cover the bottom). <br/><br/>
-
            <br/><center><img src="https://static.igem.org/mediawiki/2012/5/59/7125391185_955e181f9f_c.jpg"></center><br/>
+
<h2>Making Liquid Media </h2> <br/>
-
              <center><p>We are the 2012 University of Illinois iGEM team. We are a diverse collective of undergraduate students hailing from varying disciplines of science and engineering. Of our backgrounds in bioengineering, biochemistry, molecular biology, agricultural engineering, and bio-molecular engineering, we are brought together by common goals of exploring emergent philosophies of Synthetic Biology.</p></center><br/>
+
1. Always use a 5-2.5-2.5 ratio of Tryptone to Yeast Extract to NaCl <br/>
 +
2. Add dry ingredients first, then add MilliQ water <br/>
 +
3. No Agar! <br/>
 +
4. Autoclave by total volume <br/><br/>
 +
 
 +
<h2>Making Glycerol Stock </h2> <br/>
 +
To make 400 mL of 10% glycerol you will need:<br/><br/>
 +
 
 +
-40 mL glycerol<br/>
 +
-360 mL of MilliQ water <br/><br/>
 +
 
 +
To make 400 mL of 20% glycerol you will need:<br/><br/>
 +
 
 +
-80 mL glycerol<br/>
 +
-320 mL of MilliQ water <br/><br/>
 +
 
 +
1. Use flasks and bottles before graduated cylinders (they take forever to mix!)<br/>
 +
2. Need a stir plate and a large stir bar <br/>
 +
3. Stir until mixture is homogeneous <br/>
 +
4. Don’t autoclave! <br/><br/>
 +
 
 +
<h2>Making ddH2O</h2> <br/>
 +
- Use the small bottles. <br/>
 +
- Autoclave water by total volume. <br/><br/>
 +
 
 +
<h2>Autoclaving </h2><br/>
 +
- Robert is the source of official help on all things about the Autoclave. <br/><br/>
 +
1. Use the autoclave tape. <br/>
 +
2. Look at the Betastar for settings. <br/>
 +
3. Go by materials and total volume. <br/>
 +
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/>
 +
 
 +
<h2>Ligation </h2><br/>
 +
We used the <a href="http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf">Ginkgo bioworks protocol</a> <br/><br/>
 +
1. Control: <br/><br/>
 +
- 2 uL of Circular PSB1C3 plasmid 13 uL ddH2O <br/>
 +
- 2 uL T4 ligase buffer<br/>
 +
- 1 uL T4 ligase <br/><br/>
 +
2. P&C: <br/><br/>
 +
- 2 uL of Circular PSB1C3 plasmid<br/>
 +
- 2 uL of PUF, 11 uL ddH2O<br/>
 +
- 2 uL T4 ligase buffer<br/>
 +
- 1 uL T4 ligase<br/><br/>
 +
3. L&P:<br/><br/>
 +
- 2 uL of linear PSB1C3 plasmid<br/>
 +
- 2 uL of PUF<br/>
 +
- 11 uL ddH2O<br/>
 +
- 2 uL T4 ligase buffer<br/>
 +
- 1 uL T4 ligase<br/><br/>
<div id="prot1" style="display:none">
<div id="prot1" style="display:none">

Revision as of 19:54, 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
  • 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"