Team:UIUC-Illinois/Notebook/Protocols/Bootcamp

From 2012.igem.org

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<center><h1>Bootcamp Protocols</h1></center>
<center><h1>Bootcamp Protocols</h1></center>
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<h2>Making LB for plates</h2>
+
<h2>Making LB for plates</h2> <br/>
-
  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/><br/>
+
1. Always use a 10:5:5 ratio of Tryptone to Yeast Extract to NaCl. <br/>
-
2. Add dry ingredients first. <br/><br/>
+
2. Add dry ingredients first. <br/>
-
3. Use a 2L flask. <br/>
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3. Use a 2L flask. <br/> <br/>
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- Add the following <br/>
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4. Add the following <br/><br/>
- 10 g Tryptone <br/>  
- 10 g Tryptone <br/>  
- 5 g Yeast Extract <br/>
- 5 g Yeast Extract <br/>
- 5 g NaCl <br/>
- 5 g NaCl <br/>
-
- 1.5 g Agar (NOT agarose!) <br/><br/>
+
- 1.5 g Agar (NOT agarose!)<br/><br/>
-
4. Then add 1 L of MilliQ water. <br/><br/>
+
4. Then add 1 L of MilliQ water. <br/>
-
5. Autoclave by total volume. <br/><br/>
+
5. Autoclave by total volume.<br/>
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>
+
<h2>Making Liquid Media </h2> <br/>
-
Always use a 5-2.5-2.5 ratio of Tryptone to Yeast Extract to NaCl  
+
1. Always use a 5-2.5-2.5 ratio of Tryptone to Yeast Extract to NaCl <br/>
-
Add dry ingredients first, then add MilliQ water  
+
2. Add dry ingredients first, then add MilliQ water <br/>
-
No Agar!  
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3. No Agar! <br/>
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Autoclave by total volume  
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4. Autoclave by total volume <br/><br/>
-
<h2>Making Glycerol Stock </h2>
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<h2>Making Glycerol Stock </h2> <br/>
-
Use flasks and bottles before graduated cylinders (they take forever to mix!)
+
To make 400 mL of 10% glycerol you will need:<br/><br/>
-
Need a stir plate and a large stir bar
+
-
400 mL of 10% glycerol: 40 mL glycerol, 360 mL of MilliQ water
+
-
400 mL of 20% glycerol: 80 mL glycerol, 320 mL of MilliQ water
+
-
Don’t autoclave!
+
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<h2>Making ddH2O</h2>
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-40 mL glycerol<br/>
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Use the small bottles. Autoclave water by total volume.
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-360 mL of MilliQ water <br/><br/>
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<h2>Autoclaving </h2>
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To make 400 mL of 20% glycerol you will need:<br/><br/>
-
Robert is the source of official help on all things about the Autoclave.
+
-
Look at the Betastar for settings. Go by materials and total volume.
+
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Use the magical tape that changes color.
+
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<h2>PCR Troubleshooting</h2>
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-80 mL glycerol<br/>  
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Ran a diagnostic test to optimize buffer and temperature for the PCR. After doing the PCR (it’s nice to do this in linked PCR tubes) we run on a gel to test which worked
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-320 mL of MilliQ water <br/><br/>
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Buffers tested: GC + DMSO, HF + DMSO, GC, buffer G supermix + DMSO (3 tubes of each)
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Made a master mix of primers, phusion, template DNA, and dNTPs. This was then used this for all the tubes of GC+DMSO, HF+DMSO, and GC.
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Master Mix: 10 uL dNTP’s, 2 uL forward primers, 2 uL reverse primers, 2 uL template DNA, 1 uL phusion
+
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GC + DMSO: 18 uL H2O, 6 uL GC buffer, 5.1 uL of master mix, 0.9 uL DMSO, now divide into 3 tubes (10 uL into each PCR tube)
+
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HF + DMSO: 18 uL H2O, 6 uL HF buffer, 5.1 uL of master mix, 0.9 uL DMSO, now divide into 3 tubes (10 uL into each PCR tube)
+
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GC: 18.9 uL H2O, 6 uL GC buffer, 5.1 uL master mix, now divide into 3 tubes (10 uL into each PCR tube)
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Supermix: 15 uL buffer G, 12 uL H2O, 0.9 uL DMSO, 0.6 uL forward primer, 0.6 uL reverse primer, 0.6 uL template DNA, 0.3 uL phusion
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PCR program:
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1. 98 degrees Celsius for 3 minutes
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2. 98 degrees C for 15 seconds
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3. Temperature gradient 48-55-63 degrees for 30 seconds
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4. 72 degrees C for 1 minute
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5. Go to step 2, repeat 30 times
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6. 72 degrees C for 5 minutes
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Gel results: Only buffer G +DMSO works and it works at all temperatures.
+
 +
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>Optomized PCR</h2>
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<h2>Making ddH2O</h2> <br/>
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Worked using buffer G +DMSO supermix at 63o C.  
+
- Use the small bottles. <br/>
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Supermix (for three reactions. 10uL/rxn): 15 uL buffer G, 12 uL H2O, 0.9 uL DMSO, 0.6 uL forward primer, 0.6 uL reverse primer, 0.6 uL template DNA, 0.3 uL phusion
+
- Autoclave water by total volume. <br/><br/>
-
PCR program (running time of just under 2 hours):
+
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1. 98 degrees Celsius for 3 minutes
+
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2. 98 degrees C for 15 seconds
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3. 63 degrees C for 30 seconds
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4. 72 degrees C for 1 minute
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5. Go to step 2, repeat 30 times
+
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6. 72 degrees C for 5 minutes
+
 +
<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/>
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<h2>Ligation </h2>
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<h2>Ligation </h2><br/>
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Used the Ginkgo bioworks protocol  
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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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(Control: 2 uL of Circular PSB1C3 plasmid, 13 uL ddH2O, 2 uL T4 ligase buffer, 1 uL T4 ligase  
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1. Control: <br/><br/>
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P&C: 2 uL of Circular PSB1C3 plasmid, 2 uL of PUF, 11 uL ddH2O, 2 uL T4 ligase buffer, 1 uL T4 ligase
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- 2 uL of Circular PSB1C3 plasmid 13 uL ddH2O <br/>
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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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- 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

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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