In this course you will learn:

• The basic concepts of the standardpolymerase chain reaction (PCR) technique.

• The criteria required to design aDNA primer for PCR.

• The onlineprograms we usually useto 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 thePCR primer design criteria and how they affect the primer sensitivity andstabilityincluding; primer length, primermelting temperature, primerannealing 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 findtips for successful gelelectrophoresis. It includesall the possible problems you may encounter, and suggest you a solution for each problem.