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From 2013.igem.org

Materials

• Prepare master mix: (Total volume 50 µL)
  • 5X Rxn Buffer 10 µL
  • Oligo 1 (5µM) 7.5 µL
  • Oligo 2 (5µM) 7.5 µL
  • dNTP mix 1 µL
  • DMSO 1 µL
  • ddH2O 22 µL
  • Phusion 0.5 µL

• NOTE: you will need 10µl of the master mix per colony, plus for positive and negative controls. Adjust volumes appropriately.

• Oligos 1 and 2 are the primers for the BioBrick part, VF2 and VR. Make sure to dilute the concentration to 5 µM to prepare the appropriate amounts.

To prepare the cells

• If you have a liquid culture:
• Prepare as many PCR tubes as you have cultures you want to amplify. Add 100 µL of ddH2O to each of those PCR tubes.
• Take 3 µL from each culture you want to PCR amplify and add to the PCR tubes (make sure to properly label each tube).

• If you have plated colonies:
• Prepare a sterile environment (wipe down counter with ethanol, get out a bunsen burner and light with a striker).
• Sterilize metal innoculating loop with bunsen burner and scoop up one colony and mix into the appropriate PCR tube.

Procedure

• Prepare connected PCR tubes (one for each colony, one for positive control, and one for negative control)
• Add 10 µL master mix into each tube
• Immediately cap the negative control to prevent cross-contamination.
• Add 1 µL of diluted cell culture to its corresponding PCR tube (make sure to label everything)
• Use a positive control that you know will work and has a known part
• Close all caps and place in PCR Machine
• Run PCR program COLONPCR_Protocol

• Once this is complete, you will be able to analyze these colonies using gel electrophoresis and determine which colonies have the appropriate parts.