## Ligation Of Dna Fragments

Once the digested vectors and insert DNA are ready, ligation can be carried out. To achieve an optimal ligation, the ratio of vector to insert DNA (1:1, 1:2, 1:3 and 3:1 molar ratios) should be optimized using a small-scale ligation. The following reaction is standard for sticky end ligation of a 3.2-kb plasmid vector and a 3.0-Kb insert DNA.

1. Calculate the molar weights of the vector and insert DNA: 1 M plasmid vector = 3.2 x 1000 x 660 = 2.112 x106

2. Calculate the molar ratio of the vector to insert DNA:

 Vector DNA:insert DNA Vector Insert 1:1 1 mg 0.792 mg 1:2 1 mg 1.584 mg 1:3 1 mg 2.376 mg 3:1 1 mg 0.264 mg 3. Set up the following ligation on ice: Ligation reactions Components 1 (1:1) 2 (1:2) 3 (1:3) 4 (3:1) Vector DNA 1 mg 1 mg 1 mg 1 mg Insert DNA 0.792 mg 1.584 mg 2.376 mg 0.244 mg 10X Ligase buffer 1 ml 1 ml 1 ml 1 ml T4 DNA ligase (Weiss units) 5 units 5 units 5 units 5 units Add dd.H2O to 10 ml 10 ml 10 ml 10 ml

4. Incubate the reactions at 25°C or room temperature for 3 h for sticky-end ligation; incubate at 16°C for 6 h to overnight or 4°C 12 to 20 h for blunt-end ligation.

5. Determine the efficiency of the ligations by 1% agarose electrophoresis. When the electrophoresis is complete, take a picture of the gel stained with EtBr under UV light. As compared with unligated vector or insert DNA, high efficiency of ligation should visualize less than approximately 20% of unligated vector and insert DNA by estimation of the intensity of fluorescence. Approximately 80% of the vector and insert DNA are ligated to each other and show strong bands with molecular weight shifts. By comparing the efficiency of ligations using different molar ratios, the optimal concentration of DNA can be used as a guide for large-scale ligation.

6. Carry out large-scale ligation of the vector and insert DNA based on the optimal conditions determined by small-scale ligations. For example, if a 1:2 molar ratio of vector DNA:insert DNA is used as the optimal ligation condition, a large-scale ligation can be carried out as follows:

Vector DNA, 3 mg

Insert DNA, 4.75 mg

10X Ligase buffer, 3 ml

T4 DNA ligase (Weiss units), 15 to 50 units

Add dd.H2O to a final volume of 30 ml.

Incubate the ligation as described in small-scale ligation. Proceed to transformation.

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