Primer Design For Polymerase Chain Reaction-Tips & Tricks

DNA primer design, what your mentor never taught you

In this course you will learn:

What you’ll learn

  • Understand how different parameters can affect your primer specificity and sensitivity.
  • Understand the basics of Polymerase chain reaction in detail.
  • Learn how to avoid non-specific bands in your gel by following a specific guideline for DNA primer design.
  • Real example on how to design DNA primer using the required program.
  • Understand how to analyse gel electrophoresis results..

Course Content

  • Polymerase Chain Reaction –> 4 lectures • 12min.
  • PCR Primer Design Criteria –> 12 lectures • 36min.
  • Tools and Methods (How to…) –> 6 lectures • 23min.
  • Gel Electrophoresis Troubleshooting –> 2 lectures • 3min.

Primer Design For Polymerase Chain Reaction-Tips & Tricks

Requirements

In this course you will learn:

  • The basic concepts of the standard polymerase chain reaction (PCR) technique.
  • The criteria required to design a DNA primer for PCR.
  • The online programs we usually use to design a DNA primer.
  • Tips for troubleshooting gel electrophoresis results.

Polymerase chain reaction (PCR)

You will learn in this section:

  • Steps involved in the PCR (PCR      cycling): denaturation, annealing, extension
  • Components of the PCR reaction:      DNA template, DNA polymerase, dNTPs, forward primer and reverse primer.
  • PCR amplification program.
  • Exponential amplification.
  • The size difference between the      PCR amplification products of the first, second, and third cycle.

Criteria for PCR primer design

You will learn in this section a detailed explanation of the PCR primer design criteria and how they affect the primer sensitivity and stability including; primer length, primer melting temperature, primer annealing temperature, GC% of the primer, GC-clamp, cross homology and primer secondary structure.

Tools and methods 

In this section you will learn how to:

  • Retrieve a Gene Sequence form NCBI, and Determine the Exact Location for Each Exon on the Chromosome Using Graphics.
  • Compare Different mRNA Transcripts and Select One to Evaluate a Gene Expression in Novel Cells.
  • Understand Primer3 Setting.
  • Calculate Primer Self-Complementarity Score.
  • Check for Primer Cross Homology Using BLAT.
  • Evaluate Primers Depending on Delta G.

Gel Electrophoresis Troubleshooting

In this section you will find tips for successful gel electrophoresis. It includes all the possible problems you may encounter, and suggest you a solution for each problem.

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