IGEM:British Columbia/Protocols/Colony PCR

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Contents

Colony PCR

Materials

  • BioBrick PCR primers (G1004, G1005)
  • Taq polymerase
  • Buffer, Mg2+, dNTPs
  • Agar plate for indexing

Equipment

  • PCR
  • PCR tubes
  • sterile toothpick/pipet tip/innoculating loop

Method

  • Make master mix of primers and other PCR components
  • Add Taq polymerase.
  • Aliquot 10uL per PCR tube.
  • Touch toothpick/pipet tip/loop to colony, then index plate, then swirl around in PCR tube.
  • Run PCR:
    1. 94°C for 2 mins
    2. 94°C for 30 secs
    3. 56°C for 30 secs
    4. 72°C for 1 min per kb of expected product, rounded up to nearest kb
    5. Repeat steps 2-4 24 times.
    6. 72°C for 3 times longer then step 4 (i.e for a expected product of 1.8 kb, step 4 will be 2 min long, step 6 will be 3*2 = 6 min long
    7. 4°C hold
  • Verify PCR products on an agarose gel.
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