Endy:DNA ligation using T4 DNA ligase

Quick Ligase ligation

Materials

• 2x Quick ligase buffer (in 40µl aliquots; these are 1-time use since freeze-thaw cycles degrade the ATP in the buffer).
• ddH2O
• Purified, linearized vector (likely in H₂O or EB)
• Purified, linearized insert (likely in H₂O or EB)Italic text

Procedure

For 10µl reaction

Larger volumes can be scaled up if needed

• 5 μL 2X Quick ligase buffer
• 6:1 Molar ratio of insert to vector (~10ng vector). Try to keep total DNA concentration <100ng/rxn for optimal efficiency.
• Add (4.5 - vector and insert volume)μl ddH₂O

Method

1. Add appropriate amount of deionized H₂O to sterile PCR tube
2. Add 5 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.
3. Add appropriate amount of insert to the tube.
4. Add appropriate amount of vector to the tube.
5. 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.
6. Let the 10 μL solution sit at 22.5°C for 30 mins
7. Denature the ligase at 65°C for 10min
8. Dialyze for 20 minutes if electroporating
9. Use disks shiny side up
10. Store at -20°C

T4 ligase ligation

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)Italic text

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

1. Add appropriate amount of deionized H₂O to sterile PCR tube
2. Add in appropriate amounts of vector and insert. Heat the mixture to 42*C for 2min to free up sticky ends (can set up a thermocycler for this).
3. Add 1 μL ligation buffer to the tube.
   Pipette buffer up and down 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.
4. Add 0.5 μL ligase. Pipette mixture up and down to ENSURE MIXING OF THE ENZYME.
   Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. Just touch your tip to the surface of the liquid when pipetting to ensure accurate volume transfer.
5. Let the 10 μL solution sit at 22.5°C for 30 mins
6. Denature the ligase at 65°C for 10min
7. 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.)