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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- blah blah
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Gibson assembly
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- whreee
- blah blah
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- blah blah
- blah blah
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PCR purification
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- Used protocol and kits provided by Qiagen
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