Team:Caltech/Project/Methods and Methods

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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