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  Section: Biotechnology Methods » Molecular Biology
 
 
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Polymerase Chain Reaction

 
     
 
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

Contamination of reactions is an often encounted problem with PCR. It is best avoided by using dedicated reagents, Gilsons, and plugged tips. Setting up a PCR reaction in a different location to where template or products are worked with is also a good precaution.


Procedure

  1. DNA template: Between 1 and 5 ng of cloned DNA or between 40 and 100 ng of genomic DNA should be used per reaction. It is convenient to dilute template stocks to an appropriate concentration, e.g., 5 ng/µL in
    dH2O for cloned DNA.
  2. Primers: Primers should be prepared by butanol extraction and resuspended in dH2O at 100 ng/µL. Each primer should be used at ~100 ng per reaction.
  3. Buffer: Buffer should be prepared as a
    10X stock. 10X PCR buffer: 100 mM Tris. HCl pH 8.3, 500 mM KCl, 15 mM MgCl2. This buffer can be prepared containing 0.1% gelatin.
  4. Taq DNA polymerase: Taq should be used at 2.5 U per reaction. Amplitaq Polymerase (Perkin Elmer Cetus) is provided at 5 U/µL.
  5. Magnesium: Extra magnesium can be added to the PCR reaction. If using the buffer above, a final Mg2+ concentration of 1.5 mM will be obtained. If necessary, magnesium can be titrated to obtain an optimal concentration. Suggested concentrations for this would be 1.5, 3.0, 4.5, 6.0 and 10 mM. Magnesium can be prepared as MgCl2 at 25 mM and autoclaved. Increasing the magnesium concentration has the same effect as lowering the annealing temperature.
  6. Nucleotides: dNTPs should be prepared from 100-mM commercial stocks (Promega or Boehringer Mannheim) as a 10X stock at 2 mM each dNTP. This is most easily done by adding 2 mL of each dNTP to 92 mL dH2O in an eppendorf.
  7. Water: Water should be autoclaved and used solely for PCR. Milli-Q water is fine for PCR or “water for injection” if the distilled water is in doubt. It can be aliquotted into 1-mL volumes and kept separate from DNA and other sources of contamination. Each aliquot should be discarded following a single use.
  8. Paraffin oil: In some instruments, paraffin oil must be added to prevent evaporation of the sample.

 
     
 
 
     




     
 
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