Team:Caltech/Colony PCR

Colony PCR

Materials

• Autoclaved water
• PCR tubes
• LB broth & plates
• Qiagen PCR Core Kit: dNTPs, Taq, Buffers
• Forward & reverse primers

Procedure

1. 85.5μL autoclaved water were added to PCR tubes - one for each colony to be amplified.
2. Inoculate the tube with cells from the desired colony. With the same toothpick, simultaneously inoculate a 5mL LB liquid culture (with antibiotic) and streak an LB-agar plate.
3. Incubate PCR tubes in a thermal cycler for 10 minutes at 100°C. Remove and cool.
4. To each tube, add: 10μL 10x PCR buffer, 2μL 10mM dNTP mix, 1μL 10μM forward primer, 1μL 10μM reverse primer, 0.5μL Taq DNA polymerase. Note: it is generally much more efficient to combine all these in a single tube and scale up for multiple reactions.
5. Replace tubes in thermal cycler and initiate CPCR program (below).
6. Analyze reactions via 1% agarose gel electrophoresis:
   1. Add 2μL NEB 6x Gel Loading Buffer to 10μL CPCR product.
   2. Load into gel. Run for 30-40 minutes at 90-100V.
   3. Stain/destain in EtBr and image using UV illumination.

Program

1. Initial denaturation: 94°C, 30s
2. 30 cycles:
   1. Denaturation: 94°C, 3 min
   2. Annealing: 54°C, 30s
   3. Elongation: 72°C, 2 min
3. Final extension: 72°C, 2 min
4. Finish: 5°C hold

Primers

• Forward: 5'-GAA TTC GCG GCC GCT TCT AGA G-3'
• Reverse: 5'-CTG CAG CGG CCG CTA CTA GTA-3'