Understand the basics of Polymerase chain reaction in detail
Understand how different parameters can affect your primer specificity and sensitivity
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.
Interest in learning
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.
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.
Who this course is for:
Biology students can take this course
5 sections • 24 lectures • 1h 13m total length
Lecture 2. Standard Amplification Program + Exponential Amplification
Lecture 3. The size difference between the PCR amplification products of the 1st
Lecture 4. List of the PCR Primer Design Criteria + Two Resources
Lecture 5. The DNA Primer Length
Lecture 7. How to Calculate Enthalpy, Entropy, and Free Energy
Lecture 8. Primer Tm-The Nearest Neighbor Method
Lecture 9. Annealing Temperature (Ta) of the DNA Primer
Lecture 10. GC-Content of the DNA Primer
Lecture 11. GC Clamp at the 3’ End of the DNA Primer
Lecture 12. Primer Secondary Structure
Lecture 13. Runs & Repeats in the primer
Lecture 14. Cross homology
Lecture 15. Primer product length
Lecture 16. Retrieve a Gene Sequence form NCBI, and Determine the Exact Location
Lecture 18. Understand Primer3 Setting + Primer Self-complementrity score
Lecture 19. Calculate Primer Self-Complementarity Score
Lecture 20. Check for Primer Cross Homology Using BLAT
Lecture 21. Evaluate Primers Depending on Delta G
Lecture 22. Low Intensity of All or Some DNA Bands
I had a passion for biology from a young age as I used to focus on reading biology books more than anything else. At school time, I used to write biology assignments in a fun and creative way, drawing pictures and comics to transform the difficult parts of biology into easy and understandable pictures. I adore creativity, it is my lifestyle.