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• Centrifuge (X mL) cell culture for 15 minutes at 5000g
• Discard supernatant and resuspend in 1 mL of cloning water
• Transfer solution into bashing bead lysis/filtration tube
• Add 6 mL of fungal/bacterial DNA binding buffer
• Vortex for 5 minutes
• Spin down for 5 minutes at 5000g
• Transfer filter to a 50 mL tube
• Put spin column in collection tube and spin in a microcentrifuge for 1 minute at 10,000g
• Add 150 mL of DNA pre-wash buffer, spin for 1 minute at 10,000g, discard flow through, and repeat once
• Add 200 uL of fungal/bacterial DNA wash buffer to column, spin for 1 minute at 10,000g, and repeat 3 more times
• Transfer column to a 1.5 mL microcentrifuge tube and add 30 uL of cloning water
• Let sit for 4 minutes, then spin for 4 minutes at 10,000 g
• Measure concentration of gDNA and label tube

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• Add the following to a single PCR tube:
• 20-21.5 uL Cloning Water (depending on type of amplicon)
• 10 uL of Betaine
• 10 uL of 5x High-Fidelity Buffer
• 4 uL DMSO
• 2.5 uL 10 uM combined forward/reverse primers
• 1 uL DNTPs
• 0.5 uL of plasmid or 2 uL of gDNA
• 0.5 uL of Phusion Polymerase (kept on ice)
• Immediately run in a thermocycler for the following times/temperatures
• Initial Denaturation 98C 30 sec
• 25-35 cycles
• 98 C 5-10 sec
• 45-72 C (at annealing temp) 10-30 sec
• 72 C 15-30 sec/(length of amplicon in kb)
• Final Extension 72 C 5-10 minutes
• Hold at 4C
• Store at 4C until use

Our iGEM team shared lab space in two different labs. Because of this, we learned two different protocols for gel electrophoresis:

Making the Gel

• Measure 1 gram of Agarose per 100 mL TAE buffer (Tris base, acetic acid and EDTA)
• We generally made gels with 75 - X mL of TAE
• Microwave in a covered erlenmeyer flask for 120 seconds
• Add 1 uL of SYBR Safe per 20 mL of TAE
• Pour into gel casing
• Remove bubbles with a pipettor
• Insert well comb
• Allow to set (~20 minutes)

Running the Gel

• Remove well comb
• Place gel in electrophoresis apparatus with the wells by the cathode (black)
• Fill apparatus with TAE buffer so that the gel is completely submerged
• Load wells with 10-60 uL of 5 (PCR product): 1 (6x Loading Dye)
• Load 5-10(?) uL of ladder into first and last wells
• Run at 125(?) V and 25(?) amps for 30(?) minutes

• Put
• Things
• Here

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