Team:Caltech/Project/Methods and Methods

From 2014.igem.org

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For each 25 uL reaction mixture:
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<b>For each 25 uL reaction mixture: </b>
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<ul><li>12.5 uL Phusion Mastermix</li>
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<ul><li> 12.5 uL Phusion Mastermix</li>
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     <li>2.5 uL primer mix (10 uM of forward and reverse primer)</li>
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     <li> 2.5 uL primer mix (10 uM of forward and reverse primer)</li>
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     <li>1 uL DNA template </li>
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     <li> 1 uL DNA template </li>
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     <li>0.75 uL DMSO (optional) </li>
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     <li> 0.75 uL DMSO (optional) </li>
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     <li>Fill to 25 uL with MilliQ water </li>  
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     <li> Fill to 25 uL with MilliQ water </li>  
</ul>
</ul>
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<b>Thermal Cycler Protocol</b>
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<ul><li></li>
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</ul>
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<ul><li></li>
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<b> Making the gel </b>
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     <li>blah blah</li>
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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>
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    <li> Microwave solution for 60-90 seconds, until agarose is completely dissolved</li>
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    <li> Add 5 uL SYBR Safe per every 50 uL of agarose solution</li>
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    <li> Pour into gel casket and add comb. Let cool for around 20-30 minutes to allow gel to set</li>
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</ul>
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<b> Lane mixtures </b>
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<ul><li> For DNA ladders, mix 0.5 uL of ladder, 1 uL loading dye, 4.5 uL MilliQ water </li>
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    <li> For DNA samples (typically PCR products), mix 2 uL of sample, 1 uL loading dye, 3 uL MilliQ water </li>
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</ul>
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<b> Running the gel </b>
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<ul><li> Fill gel box with 1x TBE buffer </li>
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    <li> Load gel, then run at 200V for 20 minutes </li>
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     <li> Image gel under UV light </li>  
</ul>
</ul>
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Revision as of 20:47, 14 July 2014



Home Team Official Team Profile Project Parts Modeling Notebook Safety Attributions
Materials and Methods
Overall Project Summary

Project Details

Materials and Methods

The Experiments

Results

Data Analysis

Conclusions

References

PCR

 For each 25 uL reaction mixture:
  • 12.5 uL Phusion Mastermix
  • 2.5 uL primer mix (10 uM of forward and reverse primer)
  • 1 uL DNA template
  • 0.75 uL DMSO (optional)
  • Fill to 25 uL with MilliQ water
Thermal Cycler Protocol

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 uL SYBR Safe per every 50 uL 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 uL of ladder, 1 uL loading dye, 4.5 uL MilliQ water
  • For DNA samples (typically PCR products), mix 2 uL of sample, 1 uL loading dye, 3 uL 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
  • blah blah
  • blah blah
  • blah blah
  • blah blah

Gibson assembly
  • whreee
  • blah blah
  • blah blah
  • blah blah
  • blah blah

PCR purification
  • Used protocol and kits provided by Qiagen

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