Standard Ligation Protocol

• T4 ligase joins the 5' phosphate and the 3'-hydroxyl groups of DOUBLE stranded DNA molecules.
• Estimate the vector and insert concentrations.
• If insert is from PCR, assume that 50% is recovered, typically 5 mg, and resuspended in 10 ml of H20.
• Same with vector, assume half is recovered in purification/precipitation and resuspend in 10-20 ml of H₂O if it is not re-suspended already.
• Plan control reactions.
• One reaction with no insert
• One reaction with no vector (if enough insert is available)
• Setup each ligation mixure in the following way (see note below if over 2 ml of vector or over 4 ml of insert required)
• 200 ng of vector DNA
• 500 ng of insert DNA
• 10X Ligase buffer (Promega #M1801)
• 1 ml T4 Ligase (Promega #M1801, 3 U/ml)
• Bring volume to 10 (or 20 see note below) ml with nuclease-free water.
• IF you are doing a blunt-end ligation, you may need to add PEG (up to 15%) to increase the efficiency.
• Mix by pipetting. Do not vortex.
• If over 2 ml of vector is needed or over 4 ml of insert, do a 20 ml reaction. Otherwise 10 ml is sufficient.
• Incubate according to the guidelines below
• Incubate sticky end ligation reactions at room temperature for 3 hours (or at 4 to 8 deg C, overnight).
• Incubate blunt-end ligation reactions at 17 deg C for 4 to 18 hours.
• Heat inactivate the ligase by placing tube in 65C water bath for 10 minutes.
• This has been shown to increase the transformation efficiency
• Electorporate using 2 ml of the ligation mixture.
• The optimal concentration is measured using the electroporation time constant (between 3 to 4.5 ml).
• The more DNA/salt present, the lower the time constant.
• Plate on selective media and incubate.