Team:Caltech/Project/Methods and Methods

From 2014.igem.org

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<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='black'; modeling.style.color='white'" onMouseOut="this.bgColor='white'; modeling.style.color='black'" bgColor=white>
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='black'; modeling.style.color='white'" onMouseOut="this.bgColor='white'; modeling.style.color='black'" bgColor=white>
-
<a id='modeling' href="https://2014.igem.org/Team:Caltech/Modeling"style="color:#000000"> Modeling</a></td>
+
<a id='modeling' href="https://2014.igem.org/Team:Caltech/TXTL"style="color:#000000"> TXTL Promoter Characterization</a></td>
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='black'; notebook.style.color='white'" onMouseOut="this.bgColor='white'; notebook.style.color='black'" bgColor=white>
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='black'; notebook.style.color='white'" onMouseOut="this.bgColor='white'; notebook.style.color='black'" bgColor=white>
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<tr>
<tr>
<td width="20%"  valign="top">  
<td width="20%"  valign="top">  
-
<a href = "https://2014.igem.org/Team:Caltech/Project">Overall Project Summary</a>
+
<a href = "https://2014.igem.org/Team:Caltech/Project">Project Overview</a>
<br><br>
<br><br>
<a href = "https://2014.igem.org/Team:Caltech/Project/Details">Project Details</a>
<a href = "https://2014.igem.org/Team:Caltech/Project/Details">Project Details</a>
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<a href = "https://2014.igem.org/Team:Caltech/Project/Experiments">The Experiments</a>
<a href = "https://2014.igem.org/Team:Caltech/Project/Experiments">The Experiments</a>
<br><br>
<br><br>
-
<a href = "https://2014.igem.org/Team:Caltech/Project/Results">Results</a>
+
<a href = "https://2014.igem.org/Team:Caltech/Project/Results">Our Results</a>
-
<br><br>
+
-
<a href = "https://2014.igem.org/Team:Caltech/Project/Data">Data Analysis</a>
+
<br><br>
<br><br>
<a href = "https://2014.igem.org/Team:Caltech/Project/Conclusions">Conclusions</a>
<a href = "https://2014.igem.org/Team:Caltech/Project/Conclusions">Conclusions</a>
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</td>
</td>
<td valign="top">
<td valign="top">
-
<b>For each 25 uL reaction mixture: </b>
+
<b>For each 25 &mu;L reaction mixture: </b>
-
<ul><li> 12.5 uL Phusion Mastermix</li>
+
<ul><li> 12.5 &mu;L Phusion Mastermix</li>
-
     <li> 2.5 uL primer mix (10 uM of forward and reverse primer)</li>
+
     <li> 2.5 &mu;L primer mix (10 &mu;M of forward and reverse primer)</li>
-
     <li> 1 uL DNA template </li>
+
     <li> 1 &mu;L DNA template </li>
-
     <li> 0.75 uL DMSO (optional) </li>
+
     <li> 0.75 &mu;L DMSO (optional) </li>
-
     <li> Fill to 25 uL with MilliQ water </li>  
+
     <li> Fill to 25 &mu;L with MilliQ water </li>  
</ul>
</ul>
<b>Thermal Cycler Protocol</b>
<b>Thermal Cycler Protocol</b>
-
<ul><li> 98C for 30 seconds </li>
+
<ul><li> 98&deg;C for 30 seconds </li>
-
     <li> 98C for 10 seconds </li>
+
     <li> 98&deg;C for 10 seconds </li>
-
     <li> 53C for 30 seconds </li>
+
     <li> 53&deg;C for 30 seconds </li>
-
     <li> 72C for 15<i>x</i> seconds, where <i>x</i> is the expected length of longest PCR product (in kilobases) </li>
+
     <li> 72&deg;C for 15<i>x</i> seconds, where <i>x</i> is the expected length of longest PCR product (in kilobases) </li>
     <li> Repeat above steps 29 more times </li>
     <li> Repeat above steps 29 more times </li>
-
     <li> 72C for 10 minutes </li>
+
     <li> 72&deg;C for 10 minutes </li>
-
     <li> Hold at 4C </li>
+
     <li> Hold at 4C&deg; </li>
</ul>
</ul>
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<ul><li> Make 1% agarose solution in 1x TBE buffer (typically 0.5 g agarose per 50 mL 1x TBE buffer)</li>
<ul><li> Make 1% agarose solution in 1x TBE buffer (typically 0.5 g agarose per 50 mL 1x TBE buffer)</li>
     <li> Microwave solution for 60-90 seconds, until agarose is completely dissolved</li>
     <li> Microwave solution for 60-90 seconds, until agarose is completely dissolved</li>
-
     <li> Add 5 uL SYBR Safe per every 50 uL of agarose solution</li>
+
     <li> Add 5 &mu;L SYBR Safe per every 50 &mu;L of agarose solution</li>
     <li> Pour into gel casket and add comb. Let cool for around 20-30 minutes to allow gel to set</li>
     <li> Pour into gel casket and add comb. Let cool for around 20-30 minutes to allow gel to set</li>
</ul>
</ul>
<b> Lane mixtures </b>
<b> Lane mixtures </b>
-
<ul><li> For DNA ladders, mix 0.5 uL of ladder, 1 uL loading dye, 4.5 uL MilliQ water </li>
+
<ul><li> For DNA ladders, mix 0.5 &mu;L of ladder, 1 &mu;L loading dye, 4.5 &mu;L MilliQ water </li>
-
     <li> For DNA samples (typically PCR products), mix 2 uL of sample, 1 uL loading dye, 3 uL MilliQ water </li>  
+
     <li> For DNA samples (typically PCR products), mix 2 &mu;L of sample, 1 &mu;L loading dye, 3 &mu;L MilliQ water </li>  
</ul>
</ul>
<b> Running the gel </b>
<b> Running the gel </b>
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<td valign="top">
<td valign="top">
<b> If using colonies grown on plates: </b>
<b> If using colonies grown on plates: </b>
-
<ul><li>Pick colonies with pipette tip and re-suspend in 10 uL of MilliQ water</li>
+
<ul><li>Pick colonies with pipette tip and re-suspend in 10 &mu;L of MilliQ water</li>
</ul>
</ul>
<b> If using liquid cultures: </b>
<b> If using liquid cultures: </b>
-
<ul><li>Add 0.5 uL of 5mL liquid culture to 10 uL of MilliQ water </li>
+
<ul><li>Add 0.5 &mu;L of 5mL liquid culture to 10 &mu;L of MilliQ water </li>
</ul>
</ul>
<b> PCR reaction mixture </b>
<b> PCR reaction mixture </b>
-
<ul><li> 5 uL Phusion Mastermix </li>
+
<ul><li> 5 &mu;L Phusion Mastermix </li>
-
     <li> 0.5 uL forward primer (10 uM)</li>
+
     <li> 0.5 &mu;L forward primer (10 &mu;M)</li>
-
     <li> 0.5 uL reverse primer (10 uM)</li>
+
     <li> 0.5 &mu;L reverse primer (10 &mu;M)</li>
-
     <li> 4 uL MilliQ water</li>
+
     <li> 4 &mu;L MilliQ water</li>
-
     <li> Make 10<i>x</i> of this reaction mixture, where <i>x</i> is the number of colonies picked </li>
+
     <li> Make 10<i>x</i> &mu;L of this reaction mixture, where <i>x</i> is the number of colonies picked </li>
-
     <li> For each colony suspension, add 1 uL of the suspension to 10 uL of the PCR reaction mix </li>
+
     <li> For each colony suspension, add 1 &mu;L of the suspension to 10 &mu;L of the PCR reaction mix </li>
</ul>
</ul>
<b> Thermal Cycler Protocol </b>
<b> Thermal Cycler Protocol </b>
-
<ul><li> Lid temperature 105C </li>
+
<ul><li> Lid temperature 105&deg;C </li>
-
     <li> 98C for 10 minutes </li>
+
     <li> 98&deg;C for 10 minutes </li>
-
     <li> 98C for 30 seconds </li>
+
     <li> 98&deg;C for 30 seconds </li>
-
     <li> 53C for 15 seconds </li>
+
     <li> 53&deg;C for 15 seconds </li>
-
     <li> 72C for 15<i>x</i> seconds, where <i>x</i> is the expected length of PCR product (in kilobases) </li>
+
     <li> 72&deg;C for 15<i>x</i> seconds, where <i>x</i> is the expected length of PCR product (in kilobases) </li>
     <li> Repeat above steps 29 more times </li>
     <li> Repeat above steps 29 more times </li>
-
     <li> 72C for 5 minutes </li>
+
     <li> 72&deg;C for 5 minutes </li>
-
     <li> Hold at 4C </li>
+
     <li> Hold at 4&deg;C </li>
</ul>
</ul>
</td>
</td>
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</td>
</td>
<td valign="top">
<td valign="top">
-
<ul><li> Calculate equimolar amounts of each DNA fragment to be used in Gibson assembly. The length of the insert divided by length of the backbone multiplied by the mass of the backbone used (typically 0.5 or 1 ng) gives the mass of insert desired (in ng). Use the concentration of the DNA fragment to determine volume of DNA fragment to be added to the DNA mix. Fill to 2.5 or 5 uL of total DNA mix using MilliQ water</li>
+
<ul><li> Calculate equimolar amounts of each DNA fragment to be used in Gibson assembly. The length of the insert divided by length of the backbone multiplied by the mass of the backbone used (typically 0.5 or 1 ng) gives the mass of insert desired (in ng). Use the concentration of the DNA fragment to determine volume of DNA fragment to be added to the DNA mix. Fill to 2.5 or 5 &mu;L of total DNA mix using MilliQ water</li>
     <li> Add 3 times as much Gibson Mastermix as there is DNA mix</li>
     <li> Add 3 times as much Gibson Mastermix as there is DNA mix</li>
</ul>
</ul>
<b> Thermal Cycler Protocol </b>     
<b> Thermal Cycler Protocol </b>     
<ul><li> Lid temperature 105C</li>
<ul><li> Lid temperature 105C</li>
-
     <li> 50C for 1 hour</li>
+
     <li> 50&deg;C for 1 hour</li>
-
     <li> Hold at 4C </li>
+
     <li> Hold at 4&deg;C </li>
</ul>
</ul>
</td>
</td>
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<td valign="top">
<td valign="top">
<b> For JM109 cells: </b>
<b> For JM109 cells: </b>
-
<ul><li> Add 2 uL of plasmid to 20 uL of cells</li>
+
<ul><li> Add 2 &mu;L of plasmid to 20 &mu;L of cells</li>
     <li> Stir with pipette tip</li>
     <li> Stir with pipette tip</li>
-
     <li> Keep cells on ice</li>
+
     <li> Keep cells on ice for 5-10 minutes</li>
 +
    <li> Add 4 volumes of SOC medium and incubate in shaker at 37&deg;C for 1 hour. This step is optional if plating onto CARB. </li>
     <li> Plate with glass beads </li>
     <li> Plate with glass beads </li>
</ul>
</ul>
-
<b> For DH5&#945;-Z1 cells:</b>
+
<b> For DH5&alpha;-Z1 cells:</b>
-
<ul><li> Add 1 uL of plasmid to 40 uL of cells </li>
+
<ul><li> Add 1 &mu;L of plasmid to 40 &mu;L of cells </li>
     <li> Incubate on ice for 10 minutes </li>
     <li> Incubate on ice for 10 minutes </li>
-
     <li> Heat shock at 42C for 90 seconds </li>
+
     <li> Heat shock at 42&deg;C for 90 seconds </li>
-
     <li> Add 100 uL of SOC medium, then incubate in a 37C shaker for 30-90 minutes to recover </li>  
+
     <li> Add 100 &mu;L of SOC medium, then incubate in a 37&deg;C shaker for 30-90 minutes to recover </li>  
     <li> Plate with glass beads </li>
     <li> Plate with glass beads </li>
 +
</ul>
 +
</td>
 +
</tr>
 +
 +
<tr><td bgColor="#777777" colspan="2" height="1px"></td></tr>
 +
 +
<tr>
 +
<td width="25%"  valign="top">
 +
<h4>Liquid Culture Inoculation</h4>
 +
</td>
 +
<td valign="top">
 +
<ul><li> Add 5 &mu;L antibiotic to 5 mL LB media </li>
 +
    <li> Pick colony from solid culture with pipette tip and mix into liquid </li>
 +
    <li> Incubate in 37&deg;C shaker overnight</li>
</ul>
</ul>
</td>
</td>
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<ul><li> Used to purify PCR products of unwanted DNA </li>
<ul><li> Used to purify PCR products of unwanted DNA </li>
     <li> Use QIAquick PCR Purification Kit (Cat. No. 28107)</li>
     <li> Use QIAquick PCR Purification Kit (Cat. No. 28107)</li>
-
     <li> Deviation from established protocol: elute in 40 uL EB buffer</li>
+
     <li> Deviation from established protocol: elute in 40 &mu;L EB buffer</li>
</ul>
</ul>
</td>
</td>
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<ul><li> Use to extract plasmids from transformed colonies </li>
<ul><li> Use to extract plasmids from transformed colonies </li>
     <li> Use QIAprep Spin Miniprep Kit (Cat. No. 27104)</li>
     <li> Use QIAprep Spin Miniprep Kit (Cat. No. 27104)</li>
-
     <li> Deviation from established protocol: elute in 40 uL EB buffer</li>
+
     <li> Deviation from established protocol: elute in 40 &mu;L EB buffer</li>
</ul>
</ul>
</td>
</td>

Latest revision as of 21:43, 17 October 2014



Home Team Official Team Profile Project Parts TXTL Promoter Characterization Notebook Safety Attributions
Materials and Methods
Project Overview

Project Details

Materials and Methods

The Experiments

Our Results

Conclusions

References

PCR

For each 25 μL reaction mixture:
  • 12.5 μL Phusion Mastermix
  • 2.5 μL primer mix (10 μM of forward and reverse primer)
  • 1 μL DNA template
  • 0.75 μL DMSO (optional)
  • Fill to 25 μL with MilliQ water
Thermal Cycler Protocol
  • 98°C for 30 seconds
  • 98°C for 10 seconds
  • 53°C for 30 seconds
  • 72°C for 15x seconds, where x is the expected length of longest PCR product (in kilobases)
  • Repeat above steps 29 more times
  • 72°C for 10 minutes
  • Hold at 4C°

Gel electrophoresis

Making the gel
  • Make 1% agarose solution in 1x TBE buffer (typically 0.5 g agarose per 50 mL 1x TBE buffer)
  • Microwave solution for 60-90 seconds, until agarose is completely dissolved
  • Add 5 μL SYBR Safe per every 50 μL of agarose solution
  • Pour into gel casket and add comb. Let cool for around 20-30 minutes to allow gel to set
Lane mixtures
  • For DNA ladders, mix 0.5 μL of ladder, 1 μL loading dye, 4.5 μL MilliQ water
  • For DNA samples (typically PCR products), mix 2 μL of sample, 1 μL loading dye, 3 μL MilliQ water
Running the gel
  • Fill gel box with 1x TBE buffer
  • Load gel, then run at 200V for 20 minutes
  • Image gel under UV light

Colony PCR

If using colonies grown on plates:
  • Pick colonies with pipette tip and re-suspend in 10 μL of MilliQ water
If using liquid cultures:
  • Add 0.5 μL of 5mL liquid culture to 10 μL of MilliQ water
PCR reaction mixture
  • 5 μL Phusion Mastermix
  • 0.5 μL forward primer (10 μM)
  • 0.5 μL reverse primer (10 μM)
  • 4 μL MilliQ water
  • Make 10x μL of this reaction mixture, where x is the number of colonies picked
  • For each colony suspension, add 1 μL of the suspension to 10 μL of the PCR reaction mix
Thermal Cycler Protocol
  • Lid temperature 105°C
  • 98°C for 10 minutes
  • 98°C for 30 seconds
  • 53°C for 15 seconds
  • 72°C for 15x seconds, where x is the expected length of PCR product (in kilobases)
  • Repeat above steps 29 more times
  • 72°C for 5 minutes
  • Hold at 4°C

Gibson assembly

  • Calculate equimolar amounts of each DNA fragment to be used in Gibson assembly. The length of the insert divided by length of the backbone multiplied by the mass of the backbone used (typically 0.5 or 1 ng) gives the mass of insert desired (in ng). Use the concentration of the DNA fragment to determine volume of DNA fragment to be added to the DNA mix. Fill to 2.5 or 5 μL of total DNA mix using MilliQ water
  • Add 3 times as much Gibson Mastermix as there is DNA mix
Thermal Cycler Protocol
  • Lid temperature 105C
  • 50°C for 1 hour
  • Hold at 4°C

Transformations

For JM109 cells:
  • Add 2 μL of plasmid to 20 μL of cells
  • Stir with pipette tip
  • Keep cells on ice for 5-10 minutes
  • Add 4 volumes of SOC medium and incubate in shaker at 37°C for 1 hour. This step is optional if plating onto CARB.
  • Plate with glass beads
For DH5α-Z1 cells:
  • Add 1 μL of plasmid to 40 μL of cells
  • Incubate on ice for 10 minutes
  • Heat shock at 42°C for 90 seconds
  • Add 100 μL of SOC medium, then incubate in a 37°C shaker for 30-90 minutes to recover
  • Plate with glass beads

Liquid Culture Inoculation

  • Add 5 μL antibiotic to 5 mL LB media
  • Pick colony from solid culture with pipette tip and mix into liquid
  • Incubate in 37°C shaker overnight

PCR purification

  • Used to purify PCR products of unwanted DNA
  • Use QIAquick PCR Purification Kit (Cat. No. 28107)
  • Deviation from established protocol: elute in 40 μL EB buffer

Miniprep

  • Use to extract plasmids from transformed colonies
  • Use QIAprep Spin Miniprep Kit (Cat. No. 27104)
  • Deviation from established protocol: elute in 40 μL EB buffer