"

From 2013.igem.org

> iGEM Home

Colony PCR

Supplies needed:

PCR tubes

BioBrick PCR primers (VF2, VR)

Taq polymerase

10x Reaction Buffer

10mM dNTPs

dH2O

Colonies

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:

• Turn machine on

• Load samples in machine

• 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

5. Once finished, remove the PCR tubes from the machine.

6. Verify PCR products agerose gel or store PCR tubes at 4°C or -20°C.