Molecular Techniques and Methods

Polymerase Chain Reaction

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION




MATERIALS AND SOLUTIONS

10 mM dNTPs Mix


Taq DNA Polymerase


10 X PCR Buffer


2 uM PCR Primers


0.2-ml Sterile PCR Tubes




PROCEDURES

1. In a pre-PCR area, assemble and label the PCR (0.5 ml) reactions as follow:
  • Include the assay blank (H2O) and negative and positive controls.
  • Make a master mix and distribute to each PCR tubes.

    Component
    Volume
    Final Concentration
    10 x Buffer
    2 ul
    1 x Buffer
    25 mM MgCl2
    1.6-2 ul
    2-2.5 mM MgCl2
    2 uM Primer 1
    2 ul
    0.2 uM
    2 uM Primer 2
    2 ul
    0.2 uM
    10 mM dNTPs Mix
    0.2-0.4 ul
    100-200 uM
    DNA Template
    1-2 ul
    1-10 ng
    Taq DNA Polymerase
    0.1-0.2 ul
    0.5 U
    Distilled H2O
    -
    Final Volume
    20 ul
    20 ul


    2. Do PCR reaction as follow:

    1 cycle
    Denaturation
    95oC
    2 min
    25-40 cycles
    Denaturation
    95oC
    10 sec
    Annealing
    Tm-5oC
    15 sec
    Extension
    72oC
    1 min/kb
    1 cycle
    Final Extension
    72oC
    5-10 min


  • Tm of PCR Primer = [2 x (A+T)] + [4 x (C+G)]




    NOTES




    KIT INFORMATION




    REFERENCES



  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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