- DNA vector
- DNA insert
- T4 DNA ligase
- ligase buffer
- assorted pipetmen
- assorted pipet tips
- 0.5 ml microfuge tube
- bench top microfuge
- Place the appropriate ligase buffer, DNA vector, and DNA insert to be ligated together on ice. Label a 0.5 ml microfuge tube accordingly and place on ice.
- Add the following components to the microfuge tube in the order listed with a L20 pipetman:
|nanopure water||(17.0 – x - y) µl|
|DNA vector||x µl1|
|DNA insert||y µl1|
|ligase buffer||2.0 µl|
|T4 DNA ligase||1.0 µl|
|total volume||20.0 µl|
- Mix the contents of the tube by flicking or pipetting the solution up and down with the L20 pipetman. Place the tube in the benchtop microfuge and pulse briefly to collect the volume at the bottom of the tube.
- Leave the tube on your bench top for 10 minutes to 2 hours, depending on the nature of the ligation reaction. For cohesive (sticky) ends, the ligation reaction can proceed for only 10 minutes; however, for a blunt-ended ligation, the ligation reaction should be allowed to proceed for at least 2 hours prior to transforming.
- You may continue with transforming DNA from this ligation reaction into E. coli through electroporation. Alternatively, the ligation reaction may be stored in the -20C freezer until ready for use.
When making freezer stocks it is important to confirm the identity of your culture or plasmid and take extra precaution to not contaminate your samples during this process as this will be your only source of the genetic material or strain. Freezer stocks are made up in 15% final glycerol solutions.