DNA ligation

Endy lab protocol

Ligation Mix

Example - 10ul mix

• 1.0 μL 10X T4 ligase buffer
• 0.5 μL T4 Ligase
• 6:1 Molar ratio of insert to vector (~10ng vector)
• X μL ddH₂O to bring the total to 10μL

Calculating Insert and Vector Amounts

[math]\displaystyle{ \rm{Insert\ Mass} = 6\times\left[\frac{\rm{Insert\ Length}}{\rm{Vector\ Length}}\right]\times \rm{Vector\ Mass} }[/math]

Procedure

• Let the 10 μL solution sit at 22.5°C for 30 mins
• Denature the ligase at 65°C for 10min
• Dialyze for 20 minutes if electroporating
• Use disks shiny side up
• Store at -20°C

Knight lab protocol

Materials

• We use the Quick Ligation Kit from NEB
• Deionized, sterile H₂O
• Purified, linearized vector (in H₂O)
• Purified, linearized insert (in H₂O)

Ligation Mix

This is the same protocol that NEB recommends.

• X μL vector (equivalent to 50 ng)
• Y μL insert (3-fold molar excess, see below)
• 10 μL 2X Ligase Buffer
• (10 - X - Y) μL deionized H₂O
• 1 μL Quick Ligase

Calculating Insert Amount

[math]\displaystyle{ \rm{Insert\ Mass\ in\ ng} = 3\times\left[\frac{\rm{Insert\ Length\ in\ bp}}{\rm{Vector\ Length\ in\ bp}}\right]\times \rm{Vector\ Mass\ in\ ng} }[/math]

Procedure

1. Add appropriate amount of deionized H₂O to sterile 0.6 mL tube
2. Add 10 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. It is recommended that you aliquot the Ligation Buffer into smaller quantities.
3. Add appropriate amount of insert to the tube.
4. Add appropriate amount of vector to the tube.
5. Add 1 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. Incubate 5 mins on the benchtop.
7. Place on ice until transformation.
8. Generally 1 μL of ligation mix is sufficient for either chemical transformation or electroporation. The amount of salt in 1 μL ligation mix should not cause arcing.
9. Optional Heat-inactivate by incubating at 65°C for 20 mins. Then do a purification step to remove PEG (see notes below).

Notes

1. If you are having trouble with your ligation, there is an FAQ to help.
2. Prior to the ligation, some heat their DNA slightly (maybe ~37°C) to melt any sticky ends which may have annealed improperly at low temperatures.
3. Tom has read that ligase can inhibit transformation. By heat-inactivating the ligase, this inhibition can be avoided. However, according to the NEB FAQ, heat-inactivation of PEG (which is present in the ligation reaction) also inhibits transformation, therefore a spin-column purification is recommended prior to transformation.