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  Section: Biotechnology Methods » Molecular Biology
 
 
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Ligation of Insert DNA to Vector DNA

 
     
 
Content
Molecular Biology
  The Central Dogma
  Protein Synthesis in Cell Free Systems
  Chromosomes
  Polytene Chromosomes of Dipterans
  Salivary Gland Preparation (Squash Technique)
  Extraction of Chromatin
  Chromatin Electrophoresis
  Extraction and Electrophoresis of Histones
  Karyotype Analysis
  In Situ Hybridization
  Culturing Peripheral Blood Lymphocytes
  Microslide Preparation of Metaphases for In-Situ Hybridization
  Staining Chromosomes (G-Banding)
  Nucleic Acids
  Extraction of DNA from Bovine Spleen
  Purification of DNA
  Characterization of DNA
  DNA-Dische Diphenylamine Determination
  Melting Point Determination
  CsCl-Density Separation of DNA
  Phenol Extraction of rRNA (Rat liver)
  Spectrophotometric Analysis of rRNA
  Determination of Amount of RNA by the Orcinol Method
  Sucrose Density Fractionation
  Nucleotide Composition of RNA
  Isolation of Genomic DNA—DNA Extraction Procedure
  Isolation of Genomic DNA from Bacterial Cells
  Preparation of Genomic DNA from Bacteria
  Extraction of Genomic DNA from Plant Source
  Extraction of DNA from Goat Liver
  Isolation of Cotton Genomic DNA from Leaf Tissue
  Arabidopsis Thaliana DNA Isolation
  Plant DNA Extraction
  Phenol/Chloroform Extraction of DNA
  Ethanol Precipitation of DNA
  Isolation of Mitochondrial DNA
  Isolation of Chloroplast DNA
  DNA Extraction of Rhizobium (CsCl Method)
  Isolation of Plasmids
  RNA Isolation
  Preparation of Vanadyl-Ribonucleoside Complexes that Inhibit Ribonuclease Activity
  RNA Extraction Method for Cotton
  Isolation of RNA from Bacteroids
  Isolation of RNA from Free-Living Rhizobia
  Estimation of DNA purity and Quantification
  Fungal DNA Isolation
  Methylene Blue DNA Staining
  Transformation
  Blotting Techniques—Southern, Northern, Western Blotting
  Preparing the Probe
  Southern Blotting (First Method)
  Southern Blotting (Second Method)
  Western Blotting
  Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies
  Southern Blot
  Southern Analysis of Mouse Toe/Tail DNA
  Northern Blotting
  Restriction Digestion Methods—Restriction Enzyme Digests
  Restriction Digestion of Plasmid, Cosmid, and Phage DNAs
  Manual Method of Restriction Digestion of Human DNA
  Preparation of High-Molecular-Weight Human DNA Restriction Fragments in Agarose Plugs
  Restriction Enzyme Digestion of DNA
  Electroelution of DNA Fragments from Agarose into Dialysis Tubing
  Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose
  Ligation of Insert DNA to Vector DNA
  PCR Methods (Polymerase Chain Reaction)
  Polymerase Chain Reaction
  DNA Amplification by the PCR Method

Introduction
DNA cloning requires the DNA sequence of interest to be inserted in a vector DNA molecule. For this, both the vector as well as insert DNA is prepared by digestion with compatible restriction enzymes, so that the ends produced during digestion is complementary in both. When setting up ligation, it is important to consider the permutations that can occur and bias the relative concentration of DNA accordingly.

Usually a 5- to 10-fold excess of insert over the vector DNA is the norm. This ensures that enough ligated product will be produced in the right orientation.

Materials
  • Vector digest
  • Insert DNA
  • T4 DNA ligase
  • Ligation buffer: 50 mM Tris-HCI, 10 mM MgCI2, 20 mM DTT, 1 mM ATP,
  • 50/mg/mL
  • Nuclease-free BSA, pH 7.4 (generally supplied with the enzyme)
Procedure
  1. Set up ligation in 0.5 mL microfuge tubes as follows:
  2. Voetoriligest (DHa.5).
  3. Insert DNA.
  4. Ligation buffer.
  5. Ligase.
  6. Mix well by flicking the tube.
  7. Spin briefly in a microfuge to push all the liquid to the bottom of the tube. Incubate at 16°C overnight.
  8. Stop the reaction by placing it at 70°C for 10 minutes.
  9. Run an aliquot on a minigel and verify.
Notes
The ligation buffer supplied by the vector, along with the enzyme, usually contains ATP. However, one should verify this and in case there is not ATP or if the buffer is old, it should be supplemented with ATP.

The incubation temperature in step 4 depends on the ends. In case the ends are sticky and there is a 4-base overhang, 16°C is optimum. In case of a 2-base overhang and blunt ends, the optimum temperature is 12°C and 4°C respectively. This is because to anneal shorter fragments, lower temperature is required. However, lower temperature reduces enzyme activity and enzyme concentration should be adjusted as such.

T4 DNA ligase is much more efficient than E.Coli DNA ligase, especially in blunt end ligation, and is preferred accordingly.

Most preparations of T4 DNA ligase are calibrated in Weiss units. A 0.015 Weiss unit of T4 DNA ligase will ligate 50% of Hind III fragments of bacteriophase lambda (5/-tg) in 30 minutes at 16°C. Joining of blunt-ended molecule to one another is improved greatly by the addition of noncovalent cation (150 to 200 mM NaCl) and a low concentration of PEG.


Observations
Run aliquots of ligated DNA side by side with unligated DNA to minimize and observe the difference. Being circular, ligated plasmid DNA will tend to run faster than unligated plasmid DNA.
 
     
 
 
     




     
 
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