Ligation
Lizhen Zhu
Published: 2022-09-29 DOI: 10.17504/protocols.io.bm34k8qw
Abstract
Method of plasmid construction connection (T4 DNA ligase)
Steps
system
1.
1.5µL
4.5µL
0.5µL
2µL
12.5µL
step1
2.
Set up a system as required, all operations on ice Mix ligation system with 100µL of competent and ice bath for at least 30min (thorough contact of DNA and cell, enabling DNA to get inside cell walls)
step2
3.
42˚C heat shock for 90s (competent cell swell under high temperature, cell membrane and cell wall fully contact, enabling DNA to flow inside the cell)
Ice bath for 5min (Cell shrinks and DNA is dragged through the cell membrane)
step3
4.
Pipette 200µL of full nutrient culture (in clean bench), place in a shaker at 37˚C for 1h
5.
Spread the entire sample on the plate, observe after 16h~20h
6.
