Team:iTesla-SoundBio/ColonyPCR

Colony PCR Protocol

 

Materials:

  • Plated cells with genome or plasmid of interest
  • fwd primer 10 uM working solution
  • rev primer 10 uM working solution
  • RNase-free H2O
  • 2X Q5 master mix (this has Q5 polymerase, Q5 buffer, DNTPs)
  • PCR tubes
  • Microcentrifuge tubes
  • Tris-HCl
  • Hot plate
  • Water
  • Microcentrifuge tube stand



Procedure:

  1. Fill a beaker with water and heat until boiling on the hot plate
  2. Mark colony of interest on agar plate.
  3. Pipette 10 uL of Tris-HCl into a microcentrifuge tube
  4. Tap one colony using a pipette tip and immerse the tip into the HCl
  5. Swirl the tip in the Tris-HCl
  6. Set the microcentrifuge tube in the microcentrifuge stand and boil the cells for 5 minutes
  7. Remove and set to the side until needed

 

Recipe for Q5 Master Mix PCR

 

Two 50 uL rxn for each

Regular B. subtilis (purple tubes)

Transformation 9/29 SM (Red Tubes)

18 uL RNase-free H2O

25 uL 2X Q5 Master Mix

2 uL boiled colony

2.5 uL 10 uM 16s_fwd

2.5 uL 10 uM 16s_rev

18 uL RNase-free H2O

25 uL 2X Q5 Master Mix

2 uL boiled colony

2.5 uL 10 uM 16s_fwd

2.5 uL 10 uM 16s_rev

 

Thermocycler Program:

98 C for 30 sec

30 cycles of:

98 C for 10 sec

55 C for 30 sec

72 C for 90 sec

72 C for 2 min