Ligation of DNA fragments

Aim:

To perform the ligation of linearized T-vector with DNA fragment or the ligation of any restriction enzyme digested DNA fragments using T4 DNA ligase

Principle:

The basic strategy in molecular cloning is to insert a DNA fragment of interest (a segment of DNA) into a DNA molecule (called a vector) that is capable of independent replication in a host cell. The result is a recombinant molecule composed of the DNA insert linked to vector DNA sequences. Construction of these recombinant DNA molecules is dependent on the ability to covalently seal single-stranded nicks in DNA. This process is accomplished both in vivo and in vitro by the enzyme DNA ligase. DNA ligation is the process of joining together two DNA molecules ends (either from the same or different molecules). The enzyme that joins the DNA fragments is called DNA ligases. The DNA ligase seals the nicks in DNA by the formation of the phosphodiester bond between adjacent 3’ hydroxyl and 5’ phosphate termini. The enzyme extensively used in joining DNA fragments is T4 DNA ligase. The ligase joins both cohesive ends as well as blunt-ended DNA. It is a single polypeptide with an M.W of 68,000 Dalton requiring ATP as an energy source. The maximal activity pH range is 7.5-8.0. The enzyme exhibits 40% of its activity at pH 6.9 and 65% at pH 8.3. The DNA fragment (PCR product) has an extra ‘A’ at 3’ end so that it can complementarily bind to the ‘T’ at the 5’ end of the T- vector

 5’ end of the T- vector

Materials Required: 

  • Restriction digested T-vector and PCR product (DNA)
  • T4 DNA ligase
  • Ligation buffer
  •  Nuclease-free distilled water (autoclaved)
  • Agarose
  • Gel loading dye
  • Ethidium Bromide
  • Micropipettes
  • Micro tips
  • Microfuge
  • 50x TAE buffer
  • Electrophoresis unit and power supply
  • Microwave oven/heater
  • UV transilluminator

Procedure: 

  • Three separate vials are taken and are labeled as the reaction, +ve control and –ve control.
  • 1.5µl of PCR product of DNA fragment is added to reaction and –ve control vials only.
  • 1 µl of 10X Ligation buffer is added to each of the three vials
  • 1 μl of T4 DNA Ligase (1 U) is added to the reaction and +ve control vials only.
  • 6.5 μl, 8 μl, 7.5 μl of water is added to the reaction, +ve control and –ve control vials, respectively.
  • The total volume in each of the vials is 10 μl. Incubate for 1 hr at 37ο C
  • The prepared mixtures can be analyzed in bacterial transformation in bacterial cells or they can be analyzed onto the agarose gel

 analyzed onto agarose gel table

Results and Discussion:

(make it appropriate for the DNA fragments used in your study for ligation)

More Exp,,,,….

Restriction Enzyme Digestion

Isolation of Genomic DNA from E.coli