Vector: Insert molar ratios between 1:1 and 1:10 are optimal for single insertions (up to 1:20 for short adaptors).
Use https://nebiocalculator.neb.com/#!/ligation to calculate the required molar ratio (use 1:7 to begin with)
1. Set up the following reaction in a microcentrifuge tube on ice.
(Quick Ligase should be added last)
COMPONENT
Quick Ligase Reaction Buffer (2X)* 10 uL
Vector DNA (12 kb), x uL (50 ng)
Annealed oligos (25 uM, find MW to calculate dilution, around 1:380 dilution to make 1 ng/uL from annealed stock), 1 uL
Nuclease-free Water, up to 20 uL
Quick Ligase, 1 uL
*The Quick Ligase Reaction Buffer should be thawed and resuspended at room temperature.
2. Gently mix the reaction by pipetting up and down and microfuge briefly.
3. Incubate at room temperature (25°C) for 5 minutes.