Endy:DNA ligation using T4 DNA ligase
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Materials
- T4 DNA Ligase
- 10x T4 DNA Ligase Buffer
- Deionized, sterile H2O
- Purified, linearized vector (likely in H2O or EB)
- Purified, linearized insert (likely in H2O or EB)
Procedure
10μl Ligation Mix
Larger ligation mixes are also commonly used
- 1.0 μL 10X T4 ligase buffer
- 6:1 Molar ratio of insert to vector (~10ng vector)
- Add (8.5 - vector and insert volume)μl ddH2O
- 0.5 μL T4 Ligase
Calculating Insert Amount
[math]\displaystyle{ {Insert\ Mass\ in\ ng} = 6\times\left[\frac{{Insert\ Length\ in\ bp}}{{Vector\ Length\ in\ bp}}\right]\times{Vector\ Mass\ in\ ng} }[/math]
Method
- Add appropriate amount of deionized H2O to sterile 0.6 mL tube
- Add 1 μL ligation buffer to the tube.
Vortex buffer before pipetting to ensure that it is well-mixed.
Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation. - Add appropriate amount of insert to the tube.
- Add appropriate amount of vector to the tube.
- Add 0.5 μL ligase.
Vortex ligase before pipetting to ensure that it is well-mixed.
Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 μL, just touch your tip to the surface of the liquid when pipetting. - Let the 10 μL solution sit at 22.5°C for 30 mins
- Denature the ligase at 65°C for 20min (NEB)
- Dialyze for 20 minutes if electroporating
- Use disks shiny side up
- Store at -20°C
Notes
Make sure the buffer is completely melted and dissolved. Precipitate is DTT (or BSA?). Probably best to aliquot this buffer into smaller portions, to reduce the freeze/thaw cycles. In general, make sure the buffer still smells strongly like "wet dog" (Checking if the DTT is still good.)