Team:UNIPV-Pavia/Protocols/Ligation

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

Ligation (estimated time: 20 min + 12-16 hours overnight incubation) Materials needed:


 * Roche T4 Ligase
 * 10X Roche T4 Ligase Buffer
 * ddH2O
 * (For every ligation)
 * Add 50 ng of vector
 * Add [[Image:pv_formula_lig.png]]
 * Heat DNA mix at 65°C for 5 min for DNA denaturation
 * Add 1 µl of T4 Ligase buffer
 * Add 1 µl of T4 Ligase
 * 10 µl final volume
 * Incubate at 16°C overnight
 * Then, ligation can be conserved at 4°C or can be transformed
 * Before transformation you have to inactivate T4 Ligase:
 * Heat ligation at 65°C for 10 min.