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  Section: Biotechnology Methods » Molecular Biology
 
 
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DNA Amplification by the PCR Method

 
     
 
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

To amplify the given sample of DNA using PCR.

Principle
Polymerase chain reaction (PCR) is a very simple method for in vitro DNA amplification using Taq polymerase.
This technique innolves DNA synthesis in 3 simple steps.

Step 1. Denaturation of the template into single strands.
Step 2. Annealing of primers to the template.
Step 3. Extension of new DNA.


Materials
Millique water or autoclaved double-distilled water, sterile microfuge (0.2 mL), 10 X Taq polymerase assay buffer with MgCl2, or NTP mix solution, template DNA, forward and reverse primers, Taq DNA polymerase enzyme, sterile mineral oil, and a PCR machine.


Procedure

  1. Add 38 mL of sterile millique water (or autoclaved double distilled water) to a sterile microfuge.
  2. Add 5 mL of 10 X Taq polymerase assay buffer with MgCl2 to the microfuge.
  3. Add 3 mL of 2.5 mm dNTP mixed solution to the microfuge.
  4. Add 1 mL of control template DNA.
  5. Add 1 mol each of forward and reverse primers.
  6. Add 1–2 units (0.5–0.7 mL) of Taq DNA polymerase.
  7. Gently mix.
  8. Layer the reaction mixture with 50 mL of mineral oil to avoid evaporation.
  9. Carry out the amplification using the following reaction conditions.
  10. Initial denaturation at 940°C for 1 min.
  11. Denaturation at 940°C for 30 sec.
  12. Annealing at 480°C for 30 sec.
  13. Extension at 720°C for 1 min.
  14. Final extension at 720°C for 2 min.
Result
The DNA was amplified.
 
     
 
 
     




     
 
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