Colony PCR

Supplies needed:

• PCR tubes
• BioBrick PCR primers (VF2, VR)
• Taq polymerase
• 10x Reaction Buffer
• 10mM dNTPs
• dH2O

Steps:

1. Make master mix of PCR components except the DNA. Keep on ice.
N=number of samples *Add about extra amount worth two samples to account for water control and pipetting error. (if regular PCR is done use ca.50-200ng template DNA and use Phusion and 10x Phusion buffer instead of Taq)
2. Aliquot 25µL per PCR tube, keep tubes on ice.
*Following steps must be done near the flame*
3. Touch toothpick/ pipet tip/ loop tp colony, then index plates, then dip in the PCR tube.
*Turn of flame*

4. Run PCR:
1. Turn machine on
2. Load samples in machine
3. Select appropriate temperatures and times
1. 95°C for 10s
2. 94°C for 30s
3. 60°C for 30s
4. 68°C for 1min per kb of expected product (round up)
5. Repeat 2-4 30times
6. 68°C for 20 mins
7. 4°C forever
4. Once finished, remove the PCR tubes from the machine.
5. Verify PCR products agerose gel or store PCR tubes at 4°C or -20°C.