Team:UIUC-Illinois/Notebook/Protocols/Bootcamp

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Line 77: Line 77:
To make 1 Liter of LB: <br/><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/>
1. Always use a 10:5:5 ratio of Tryptone to Yeast Extract to NaCl. <br/>
-
2. Add dry ingredients first. <<br/>
+
2. Add dry ingredients first. <br/>
-
3. Use a 2L flask. <br/>
+
3. Use a 2L flask. <br/> <br/>
4. Add the following <br/><br/>
4. Add the following <br/><br/>
- 10 g Tryptone <br/>  
- 10 g Tryptone <br/>  
Line 88: Line 88:
6. Pour 25 mL on each plate (just enough to cover the bottom). <br/><br/>
6. Pour 25 mL on each plate (just enough to cover the bottom). <br/><br/>
-
<h2>Making Liquid Media </h2> <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/>
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/>
2. Add dry ingredients first, then add MilliQ water <br/>
Line 122: Line 122:
<h2>Ligation </h2><br/>
<h2>Ligation </h2><br/>
-
- We used the <a href="http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf">Ginkgo bioworks protocol</a> <br/><br/>
+
We used the <a href="http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf">Ginkgo bioworks protocol</a> <br/><br/>
-
- Control: 2 uL of Circular PSB1C3 plasmid, 13 uL ddH2O, 2 uL T4 ligase buffer, 1 uL T4 ligase<br/>
+
1. Control: <br/><br/>
-
- P&C: 2 uL of Circular PSB1C3 plasmid, 2 uL of PUF, 11 uL ddH2O, 2 uL T4 ligase buffer, 1 uL T4 ligase<br/>
+
- 2 uL of Circular PSB1C3 plasmid 13 uL ddH2O <br/>
-
- L&P: 2 uL of linear PSB1C3 plasmid, 2 uL of PUF, 11 uL ddH2O, 2 uL T4 ligase buffer, 1 uL T4 ligase<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/>

Latest revision as of 15:31, 4 June 2012

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Protocols

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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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