IGEM:British Columbia/Protocols/Colony PCR

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 (details to be worked out when we get the supplies)
• 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. 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.