Team:UIUC-Illinois/Notebook/Protocols

From 2012.igem.org

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<div id="protocoloverview">
<div id="protocoloverview">
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<center><h1>Protocol Overview</h1></center>
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<center><h1>Bootcamp Protocols</h1></center>
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<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/>
 +
 
 +
<h2>Making Liquid Media </h2> <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>
</div>

Revision as of 16:32, 4 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

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