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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.

1x (µL) x N ( µL)

dsH2O 18.35 x N 10x rxn buffer 2.5 x N 10mM dNTP 1 x N 10mM FW primer VF2 1 x N 10mM RE primer VR 1 x N DMSO 1 x N DNA - - Taq 0.15 Total 25 x N

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. 56°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.