Team:LMU-Munich/Notebook/Protocols/22 Ligation

source: OpenWetWare: http://openwetware.org/wiki/DNA_Ligation

Materials

Reagents

• 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)

Equipment

Vortex 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

{\rm Insert\ Mass\ in\ ng} = 6\times\left[\fracTemplate:\rm Insert\ Length\ in\ bpTemplate:\rm Vector\ Length\ in\ bp\right]\times{\rm Vector\ Mass\ in\ ng}

The insert to vector molar ratio can have a significant effect on the outcome of a ligation and subsequent transformation step. Molar ratios can vary from a 1:1 insert to vector molar ratio to 10:1. It may be necessary to try several ratios in parallel for best results.

Method

1. Add appropriate amount of deionized H2O to sterile 0.6 mL tube 2. 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. 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 0.5 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