85.5μL autoclaved water were added to PCR tubes - one for each colony to be amplified.
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.
Incubate PCR tubes in a thermal cycler for 10 minutes at 100°C. Remove and cool.
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.
Replace tubes in thermal cycler and initiate CPCR program (below).
Analyze reactions via 1% agarose gel electrophoresis:
Add 2μL NEB 6x Gel Loading Buffer to 10μL CPCR product.
Load into gel. Run for 30-40 minutes at 90-100V.
Stain/destain in EtBr and image using UV illumination.
Program
Initial denaturation: 94°C, 30s
30 cycles:
Denaturation: 94°C, 3 min
Annealing: 54°C, 30s
Elongation: 72°C, 2 min
Final extension: 72°C, 2 min
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'
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