IGEM:British Columbia/Protocols/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.