Ligation of digested product

/Ligation of digested product
Ligation of digested product2018-02-23T12:23:46+00:00

Ligation of digested product

Ligation of digested product

Introduction

To perform ligation of Lambda (λ) HindIII digest and checking of the ligation reaction through agarose gel electrophoresis.
Two linear DNA molecule ends (either from the same or different molecules) can be joined together through a process called ligation. This process involves the formation of a covalent bond between two DNA fragments (having blunt or overhanging, complementary, ‘sticky’ ends) by the help of an enzyme named as ligase. DNA ligase forms a phosphodiester bond between the 3’ hydroxyl of one nucleotide and the 5’ phosphate of another. This process is the key player in constructing recombinant DNA molecule.
Recombinant DNA is made possible by two important enzymes, restriction enzymes and DNA ligase. Restriction enzymes “cut” DNA at a specific location and DNA ligase is used to “glue” two fragments of DNA together. DNA ligation is the process through which two DNA molecule ends from the same or different molecules are joined together. During this process a phosphodiester bond is formed between the 3′ hydroxyl of one fragment and the 5′ phosphate of another. This ligation reaction is catalyzed by a DNA ligase enzyme which ligates DNA fragments having blunt or overhanging, complementary, ends. It is easier to ligate molecules with complementary sticky ends than blunt ends. The commonly used DNA ligases in nucleic acid research is T4 DNA ligase and E. COLI DNA ligase. E. COLI DNA ligase is more specific for cohesive ends than T4 DNA ligase but can’t be used for cloning purpose. T4 DNA ligase is the most versatile and commonly used ligase for DNA cloning. T4 DNA ligase is approximately 60000 dalton (60 kD) protein produced by Bacteriophage T4. This ATP dependent enzyme covalently joins blunt or compatible cohesive ends, as well as nicks in double-stranded DNA. A 5′-phosphoryl group is required for ligation to a 3′-hydroxyl. Generally cohesive end ligation is carried out at lower temperature (12° C to 16° C) for the maintenance of a good balance between annealing of ends and activity of the enzyme. Blunt end ligation can be carried out at 24° C as annealing of ends is not a factor. Due to the lack of cohesive ends blunt end ligation is more complex compared to cohesive end ligation.

Requirements

Reagents

  1. Agarose
  2. DNA staining solution
  3. Electrophoresis buffer
  4. 6x Gel-loading buffer
  5. Lambda DNA Hind III digest
  6. T4 DNA ligase
  7. 10X Ligase assay buffer
  8. Mili Q water
  9. Ligation Buffer

Equipments

  1. Horizontal electrophoresis buffer
  2. Power supply

Procedure

  1. Before starting the experiment, crush ice and place the vials containing Lambda DNA-HindIII digest, 10X ligase buffer and T4 DNA ligase onto it.
  2. In this experiment Lambda DNA-HindIII digest is ligated with T4 DNA ligase.
  3. Set up the reaction mixture as follows:
  • Lambda (λ) DNA- HindIII digests: 4ul
  • 10X Ligase Assay Buffer: 1ul
  • Milli Q water: 4ul
  • T4 DNA Ligase: 1ul

Total: 10ul

  1. After preparing the reaction tube, mix the components by gentle pipetting and tapping.
  2. Incubate the tubes at 16oC waterbath for 3 hours.
  3. After incubation run the samples on agarose gel as given below.
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