Cloning with NEB Instant Sticky - end Ligase Master Mix ( M0370 )
Place master mix tube on ice and flick a few times to mix .
Combine 20–100 ng of vector with a 3 - fold molar excess of insert and q . s . to 5μl with dH2O .
Add 5 μl of Instant Sticky - end Ligase Master Mix , mix thoroughly by pipetting up and down 7 - 10 times , and place on ice .
The sample is now ready to be used for transformation .
Thaw competent cells on ice .
Aliquot 50 μl of cells into a 1.5 ml microcentrifuge tube .
Add 2 μl of the ligation reaction to the cells and mix by finger - flicking .
Do not vortex the tube .
Incubate the tube on ice for 30 minutes .
Do not mix .
Heat shock at 42°C for 30 seconds .
Return tube to ice for 2 minutes .
Add 950 μl recovery media ( e . g . SOC ) to the tube .
Incubate for one hour at 37°C with rotation or shaking ( 200–250 rpm ) .
Spread 100 μl of the outgrowth ( undiluted or diluted 1 : 5 with recovery media ) onto appropriate antibiotic selection plates and incubate overnight at 37°C .
