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Introduction

  • Function: DNA polymerase is an enzyme responsible for duplicating the DNA content of a cell during cell division.

  • Direction: DNA polymerase synthesizes DNA in the 5' to 3' direction, adding nucleotides to the 3'-OH group of the growing DNA strand.

  • PCR Use: DNA polymerase is widely used in Polymerase Chain Reaction (PCR) to amplify DNA segments.

  • Reaction Setup: Assemble all reaction components on ice and quickly transfer to a preheated thermocycler.

  • Thermocycling Conditions: Initial denaturation at 95°C for 30 seconds, followed by 30 cycles of denaturation, annealing, and extension, and a final extension at 68°C for 5 minutes.

  • Template Quality: High-quality, purified DNA templates enhance PCR success.

  • Primer Design: Primers should be 20-40 nucleotides in length with a GC content of 40-60%.

  • Mg++ Concentration: Optimal Mg++ concentration is 1.5–2.0 mM for most PCR products.

Function [1]

  • Main Role: DNA polymerase duplicates the DNA content of a cell during cell division.

  • Synthesis Direction: It synthesizes DNA in the 5' to 3' direction.

  • Template Requirement: Requires a single-stranded DNA template to copy.

  • Primer Requirement: Cannot start the new molecule itself; needs a short primer.

  • Types: Different types of DNA polymerases exist, each with specific functions.

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PCR Protocol [2]

  • Enzyme: Taq DNA Polymerase is commonly used in PCR.

  • Reaction Setup: Assemble all components on ice and transfer to a preheated thermocycler.

  • Component Volumes: Use specific volumes for 25 μl and 50 μl reactions.

  • Thermocycling: Initial denaturation at 95°C for 30 seconds, followed by 30 cycles of denaturation, annealing, and extension.

  • Final Extension: Perform a final extension at 68°C for 5 minutes.

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Reaction Setup [2]

  • Ice: Assemble all reaction components on ice.

  • Preheat: Quickly transfer reactions to a preheated thermocycler at 95°C.

  • Mixing: Gently mix the reaction and collect all liquid at the bottom of the tube.

  • Mineral Oil: Overlay the sample with mineral oil if using a PCR machine without a heated lid.

  • Transfer: Move PCR tubes from ice to the PCR machine and begin thermocycling.

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Thermocycling Conditions [2]

  • Initial Denaturation: 95°C for 30 seconds.

  • Cycles: 30 cycles of denaturation, annealing, and extension.

  • Denaturation: 95°C for 15-30 seconds.

  • Annealing: 45-68°C for 15-60 seconds.

  • Extension: 68°C for 1 minute per kb.

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Template Quality [2]

  • High Quality: Use high-quality, purified DNA templates.

  • Genomic DNA: 1 ng–1 μg for a 50 μl reaction.

  • Plasmid DNA: 1 pg–1 ng for a 50 μl reaction.

  • Enhancement: High-quality templates greatly enhance PCR success.

  • Purification: Ensure DNA templates are free from contaminants.

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Primer Design [3]

  • Length: Primers should be 20-40 nucleotides in length.

  • GC Content: Ideally have a GC content of 40-60%.

  • 3' End: Should contain a G or C to clamp the primer.

  • Complementarity: 3' ends of primers should not be complementary to each other.

  • Melting Temperature: Optimal Tm for primers is 52-58°C.

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Mg++ Concentration [2]

  • Optimal Range: 1.5–2.0 mM is optimal for most PCR products.

  • Standard Buffer: Final Mg++ concentration in 1X Standard Taq Reaction Buffer is 1.5 mM.

  • Optimization: Mg++ can be optimized in 0.5 or 1.0 mM increments using MgCl2.

  • Difficult Targets: Amplification of GC-rich sequences may require higher Mg++ concentration.

  • Additives: Additives like DMSO or formamide can improve amplification of difficult targets.

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Related Videos

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