Team:Caltech/Project/Methods and Methods

From 2014.igem.org

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<b>Thermal Cycler Protocol</b>
<b>Thermal Cycler Protocol</b>
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<ul><li> 98C for 30 seconds </li>
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    <li> 98C for 10 seconds </li>
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    <li> 53C for 30 seconds </li>
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    <li> 72C for 15<i>x</i> seconds, where <i>x</i> is the expected length of longest PCR product (in kilobases) </li>
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    <li> Repeat above steps 29 more times </li>
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    <li> 72C for 10 minutes </li>
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    <li> Hold at 4C </li>
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Revision as of 21:14, 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
  • 98C for 30 seconds
  • 98C for 10 seconds
  • 53C for 30 seconds
  • 72C for 15x seconds, where x is the expected length of longest PCR product (in kilobases)
  • Repeat above steps 29 more times
  • 72C 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 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

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

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

PCR purification
  • Used protocol and kits provided by Qiagen

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