DNA Ligation Protocol

Standard Ligation Reaction for DNA

A. Requirements:

1. Purification: Before it is ligated, the DNA should be purified, either with the PCR Product Purification Kit or by phenol extraction and ethanol precipitation

2. Dephosphorylation: For insertion of DNA into plasmid vectors the vector DNA should be dephosphorylated with Alkaline Phosphatase (unless it is to be recircularized)

3. Reaction volume:

• To prepare the DNA for ligation, dissolve it in 1x concentrated DNA Dilution Buffer (prepared from kit vial 2) to make a total volume of 10 μl.

• If the total volume of DNA solution in 1x DNA Dilution Buffer is greater than 10 μl, then increase the volume of all other reagents in the reaction accordingly and incubate the ligation reaction for 30 min (instead of 5 min).

4. Molar ratio:

• The molar ratio of vector DNA to insert DNA the standard ligation reaction should be 1:3, e.g. 50 ng linearized, dephosphorylated plasmid vector DNA plus 150 ng insert DNA (if the vector and insert DNA are approx. the same length).

• Alternatively, if the vector DNA and insert DNA are not similar in length, you may use a 1:1 or 1:2 molar ratio of vector to insert.

• A molar ratio of 1: 5 can be used for sticky-end ligations.

However, if a 1:5 ratio (vector:insert) is used for blunt-end ligation, the resulting product will generate fewer transformed colonies.

5. Transformation: To avoid inhibiting the transformation reaction with surplus DNA, use no more than 1/10 of the ligation reaction mixture in the transformation.

6. Maximum amount of DNA: The maximum amount of DNA to be ligated in 5 min should not exceed 200 ng.

7. T4 DNA Ligase inactivation: T4 DNA ligase can be completely inactivated by 10 min incubation at 65°C. Inactivation is necessary only if the ligation reaction mixture is to be used in experiments other than transformation assays. Heat inactivation of the ligation reaction mixture before transformation causes the number of transformed colonies to decrease drastically (> factor of 20).

B. Working Solution: Prepare 1x conc. DNA Dilution Buffer by mixing 5x conc. DNA Dilution Buffer (vial 2) thoroughly, and then diluting it fivefold with double dist. water.

C. Procedure:

1. Dissolve vector DNA and insert DNA in thoroughly mixed and diluted 1 x conc. DNA Dilution Buffer to a final volume of 10 µl in a sterile reaction vial.

2. Mix thoroughly T4 DNA Ligation Buffer. Add 10 μl T4 DNA Ligation Buffer to the reaction vial, Mix thoroughly.

3. Add 1 μl T4 DNA Ligase, Mix thoroughly.

4. Incubate for 15 min at RT.

5. The ligation reaction mixture can be used directly for the transformation of competent cells, or can be stored without heat inactivation at -15 to -25°C. Do not use more than 1/10 of the volume of the ligation reaction mixture for the transformation assay.

6. The ligated DNA can be analyzed by agarose gel electrophoresis.

DNA Ligation Protocol (Cell Biology Lab):

Requirements:

We use Roche Ligation Kit (Cat. No. 11 635 379 001)

Purification: Before it is ligated, the DNA should be purified, either with the PCR Product Purification Kit or by phenol extraction and ethanol precipitation

Dephosphorylation: For insertion of DNA into plasmid vectors the vector DNA should be dephosphorylated with Alkaline Phosphatase (unless it is to be recircularized)

Procedure

1. Dissolve vector DNA and insert DNA in thoroughly mixed and diluted 1 x conc. DNA Dilution Buffer (Vial 2) to a final volume of 10 μl in a sterile reaction vial.

2. Mix thoroughly T4 DNA Ligation Buffer (vial 1). You must always mix the contents of vial 1 immediately before using it.

3. Add 10 μl T4 DNA Ligation Buffer (vial 1) to the reaction vial.

4. Mix thoroughly.

5. Add 1 μl T4 DNA Ligase (vial 3). Mix thoroughly.

6. Incubate for 15 min at 15-25°C.

7. The ligation reaction mixture can be used directly for the transformation of competent cells, or can be stored without heat inactivation at -15 to -25°C.