Team:Caltech/Project/Methods and Methods

PCR

• 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

• 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

• 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

• 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

• 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 uL of MilliQ water

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

• 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

• 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

• 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

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

PCR purification

• Use QIAquick PCR Purification Kit (Cat. No. 28107)
• Deviation from established protocol: elute in 40 uL EB buffer