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
- 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
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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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- Calculate equimolar amounts of each DNA fragment to be used in Gibson assembly. The length of the insert divided by length of the backbone multiplied by the mass of the backbone used (typically 0.5 or 1 ng) gives the mass of insert desired (in ng). Use the concentration of the DNA fragment to determine volume of DNA fragment to be added to the DNA mix. Fill to 2.5 or 5 uL of total DNA mix using MilliQ water
- Add 3 times as much Gibson Mastermix as there is DNA mix
Thermal Cycler Protocol
- Lid temperature 105C
- 50C for 1 hour
- Hold at 4C
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Transformations
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For JM109 cells:
- Add 2 uL of plasmid to 20 uL of cells
- Stir with pipette tip
- Keep cells on ice
- Plate with glass beads
For DH5& alpha Z1 cells:
- Add 1 uL of plasmid to 40 uL of cells
- Incubate on ice for 10 minutes
- Heat shock at 42C for 90 seconds
- Add 100 uL of SOC medium, then incubate in a 37C shaker for 30-90 minutes to recover
- Plate with glass beads
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PCR purification
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- Used to purify PCR products of unwanted DNA
- Use QIAquick PCR Purification Kit (Cat. No. 28107)
- Deviation from established protocol: elute in 40 uL EB buffer
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Miniprep
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- Use to extract plasmids from transformed colonies
- Use QIAprep Spin Miniprep Kit (Cat. No. 27104)
- Deviation from established protocol: elute in 40 uL EB buffer
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