Team:UNIPV-Pavia/Protocols/Pcr

The protocols we used


 * LB medium preparation
 * Plasmid resuspension from IGEM paper spots
 * Transformation
 * Plasmid extraction
 * BioBrick digestion with restriction enzymes
 * DNA gel extraction
 * DNA precipitation with sodium acetate
 * Antarctic Phosphatase
 * Ligation
 * PCR

PCR (estimated time: 3 hours and 30 min) Materials needed:


 * MgCl2
 * Buffer
 * dNTPs
 * ddH2O
 * Taq Polymerase
 * VF2 primer
 * VR primer
 * For every DNA sample you want to amplify, put:
 * 2 µl buffer
 * 0.6 µl MgCl2
 * 0.4 µl dNTPs
 * 1 µl DNA (or ddH2O for blank sample). If you are performing a colony PCR, pick up the desired colony from a plate with a tip and dip it in the solution.
 * 0.2 µl Taq Polymerase
 * 250 nM VF2 primer
 * 250 nM VR primer
 * A proper amount of ddH2O to have 20 µl of total reaction volume
 * into an eppendorf tube.
 * Put the eppendorf tube in the thermal cycler and set this program:
 * 95°C 10 min
 * CYCLE:
 * 95°C 30 sec
 * 60°C 1 min
 * 72°C 1-3 min
 * for 35 cycles
 * 72°C 7 min
 * 16°C forever.
 * Now you can add a loading buffer to the solution and perform electrophoresis to check the amplified sequence length.