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.
