Overall Project Summary
Project Details
Materials and Methods
The Experiments
Results
Data Analysis
Conclusions
References
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PCR
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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
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Gel electrophoresis
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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
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Colony PCR
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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
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Gibson assembly
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PCR purification
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- Used protocol and kits provided by Qiagen
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