Team:Caltech/Project/Methods and Methods

PCR

• 12.5 μL Phusion Mastermix
• 2.5 μL primer mix (10 μM of forward and reverse primer)
• 1 μL DNA template
• 0.75 μL DMSO (optional)
• Fill to 25 μL with MilliQ water

• 98°C for 30 seconds
• 98°C for 10 seconds
• 53°C for 30 seconds
• 72°C for 15x seconds, where x is the expected length of longest PCR product (in kilobases)
• Repeat above steps 29 more times
• 72°C for 10 minutes
• Hold at 4C°

Gel electrophoresis

• 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 μL SYBR Safe per every 50 μL of agarose solution
• Pour into gel casket and add comb. Let cool for around 20-30 minutes to allow gel to set

• For DNA ladders, mix 0.5 μL of ladder, 1 μL loading dye, 4.5 μL MilliQ water
• For DNA samples (typically PCR products), mix 2 μL of sample, 1 μL loading dye, 3 μL MilliQ water

• Fill gel box with 1x TBE buffer
• Load gel, then run at 200V for 20 minutes
• Image gel under UV light

Colony PCR

• Pick colonies with pipette tip and re-suspend in 10 μL of MilliQ water

• Add 0.5 μL of 5mL liquid culture to 10 μL of MilliQ water

• 5 μL Phusion Mastermix
• 0.5 μL forward primer (10 μM)
• 0.5 μL reverse primer (10 μM)
• 4 μL MilliQ water
• Make 10x μL of this reaction mixture, where x is the number of colonies picked
• For each colony suspension, add 1 μL of the suspension to 10 μL of the PCR reaction mix

• Lid temperature 105°C
• 98°C for 10 minutes
• 98°C for 30 seconds
• 53°C for 15 seconds
• 72°C for 15x seconds, where x is the expected length of PCR product (in kilobases)
• Repeat above steps 29 more times
• 72°C for 5 minutes
• Hold at 4°C

Gibson assembly

• 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 μL of total DNA mix using MilliQ water
• Add 3 times as much Gibson Mastermix as there is DNA mix

• Lid temperature 105C
• 50°C for 1 hour
• Hold at 4°C

Transformations

• Add 2 μL of plasmid to 20 μL of cells
• Stir with pipette tip
• Keep cells on ice for 5-10 minutes
• Add 4 volumes of SOC medium and incubate in shaker at 37°C for 1 hour. This step is optional if plating onto CARB.
• Plate with glass beads

• Add 1 μL of plasmid to 40 μL of cells
• Incubate on ice for 10 minutes
• Heat shock at 42°C for 90 seconds
• Add 100 μL of SOC medium, then incubate in a 37°C shaker for 30-90 minutes to recover
• Plate with glass beads

Liquid Culture Inoculation

• Add 5 μL antibiotic to 5 mL LB media
• Pick colony from solid culture with pipette tip and mix into liquid
• Incubate in 37°C shaker overnight

PCR purification

• Used to purify PCR products of unwanted DNA
• Use QIAquick PCR Purification Kit (Cat. No. 28107)
• Deviation from established protocol: elute in 40 μL EB buffer

Miniprep

• Use to extract plasmids from transformed colonies
• Use QIAprep Spin Miniprep Kit (Cat. No. 27104)
• Deviation from established protocol: elute in 40 μL EB buffer