Jonesma Lab:Protocols:colony

From OpenWetWare
Jump to navigationJump to search

Colony PCR

  1. Take a colony and resuspend in 5 μL dH2O
  2. Streak the tip on a fresh antibiotic plate and incubate at 37°C to regenerate colony
  3. Add the PCR reagents to a tube
  4. Setup additional tubes as needed- always include a positive and negative control
  5. Add tubes to PCR machine, and run with program below
  6. Run PCR product out on a gel

Reagents for PCR

2 μL 10x PCR buffer

2 μL 1/4 dNTPS

2 μL 20mM MgCl2

6 μL dH2O

1 μL 10μM Forward primer

1 μL 10μM Reverse primer

1 μL Taq

PCR program

  1. 94°C 5 min
  2. 94°C 30s
  3. 55°C 30s
  4. 72°C 1min
  5. Loop to #2 for 35 cycles
  6. 72°C 2min
  7. 10°C indefinitely