Rolling circle DNA ampliphication kit

This method uses Amersham's Templiphi amplfication method to prepare DNA for sequencing

1. Overnight culture

Inoculate from glycerol stock plate into 1X LB containing appropriate antibiotic in to either culture tubes (2ml) using pipette tip or deep well block (1.5ml) using hedgehog. Use Qiagen Airpore tape to seal blocks.

Let grow 20-21 hours at 37 C on orbital shaker set at speed of 5.

2. Denature DNA

Remove from 37 C and transfer 0.2 Ð 0.5 ul of this culture into 5 ul of sample buffer. Denature in a thermocycler at 95 C for 3 minutes (using Marsh strips and caps). Cool to 4 C (rate of cooling not crucial).

3. Ampliphy DNA

Thaw enzyme mix on ice at 4C. Remove amount needed and immediately replace back on ice in fridge.

Make a master mix of 5ul reaction buffer and 0.2ul enzyme mix. MUST keep on ice. Transfer 5ul of master mix to cooled, denatured sample. Incubate in a thermocycler at 30 C for 16 hours, then 65 C for 10 minutes (to heat-inactivate the enzyme). Cool to 4 C.

Product can be viscous. Dilute product with 40 ul 10mM Tris.