Primer Design For Polymerase Chain Reaction-Tips & Tricks
- Description
- Curriculum
- FAQ
- Reviews
In this course you will learn:
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The basic concepts of the standard polymerase chain reaction (PCR) technique.
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The criteria required to design a DNA primer for PCR.
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The online programs we usually use to design a DNA primer.
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Tips for troubleshooting gel electrophoresis results.
Polymerase chain reaction (PCR)
You will learn in this section:
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Steps involved in the PCR (PCR cycling): denaturation, annealing, extension
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Components of the PCR reaction: DNA template, DNA polymerase, dNTPs, forward primer and reverse primer.
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PCR amplification program.
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Exponential amplification.
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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:
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Retrieve a Gene Sequence form NCBI, and Determine the Exact Location for Each Exon on the Chromosome Using Graphics.
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Compare Different mRNA Transcripts and Select One to Evaluate a Gene Expression in Novel Cells.
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Understand Primer3 Setting.
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Calculate Primer Self-Complementarity Score.
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Check for Primer Cross Homology Using BLAT.
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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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1
Introduction -
2
Lecture 1. PCR Cycling
This video provides an introduction to the Polymerase Chain Reaction (PCR) technique.
The video explains:
- Components of the PCR reaction: DNA template, DNA polymerase, dNTPs, forward primer and reverse primer.
- Steps involved in the PCR (PCR cycling: denaturation, annealing, and extension.
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3
Lecture 2. Standard Amplification Program + Exponential Amplification
This video explains:
The standard PCR amplification program.
The exponential amplification.
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4
Lecture 3. The size difference between the PCR amplification products of the 1st
In PCR amplification, how many dsDNA products we get, where the two strands of the dsDNA, are with the same length of the target sequence?
Let's find out in this lecture!
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5
Quiz
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Lecture 4. List of the PCR Primer Design Criteria + Two Resources
Why we need to learn how to design a DNA primer?
Have you ever got a non-specific band in your gel electrophoresis runs? Or got a band with a different size than your target DNA? Well, I bet the answer is yes. How a primer sequence can lead to this?
In this session, we are going to learn how to design a DNA primer that really work.
First, let's watch this video that:
- Lists the primer design criteria.
- Define the primer specificity and sensitivity.
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7
Lecture 5. The DNA Primer Length
How does the primer length play a role in the designing criteria?
Well, let's find out in this lecture.
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8
Lecture 6. Primer Melting Temperature (Tm)-Wallace equation, Marmmur & Doty equa
There is 4 approximation methods used to calculate the primer melting temperature (Tm), each one gives a reasonable result for a primer with a certain length.
In this lecture, we are going to explain in details, how to calculate the Tm using 3 calculation methods:
- Wallace equation.
- Marmur and Doty equation.
- Salt-adjusted method.
Keep your full attention.
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Lecture 7. How to Calculate Enthalpy, Entropy, and Free Energy
Enthalpy, entropy, and free energy are somehow difficult for the beginners to understand instantly. This lecture explains them in a very easy way...
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Lecture 8. Primer Tm-The Nearest Neighbor Method
The nearest-neighbor method is more complicated than the other melting temperature calculation methods. In this lecture we simplified the complicated parts for you.
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Lecture 9. Annealing Temperature (Ta) of the DNA Primer
The Annealing temperature (Ta), is at which the primers anneal (or bind) to the single stranded DNA and form a hydrogen bond.
This lecture will explain:
- How do we determine the annealing temperature empirically?
- What happens if the Ta is too low?
- What happens if the Ta is too high?
- What is the required Ta, for the forward primer and the reverse primer, to attach to the ssDNA at the same time?
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12
Lecture 10. GC-Content of the DNA Primer
This lecture explain
- The importance of the GC-content of the primer.
- How do we calculate the GC-content percentage?
- What is the ideal GC-content percentage of the primer?
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13
Lecture 11. GC Clamp at the 3’ End of the DNA Primer
In this lecture, you will understand:
- GC Clamp definition.
- The importance of the GC Clamp at the 3' end of the DNA primer.
- Designing criteria to be considered at the 3' end of the DNA primer.
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14
Lecture 12. Primer Secondary Structure
This lecture explains:
- The primer secondary structure definition.
- Types of the primer secondary structure (primer self-dimer, primer cross-dimer, primer hairpin).
- Guidelines must be considered to avoid the primer secondary structure.
- When do we consider the primer secondary structure as harmless.
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15
Lecture 13. Runs & Repeats in the primer
This lecture explains:
- What do we mean by a primer with runs?
- What do we mean by a primer with repeats?
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Lecture 14. Cross homology -
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Lecture 15. Primer product length
Learn how to measure the primer product length.
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Quiz- 2
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Quiz- 3
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Lecture 16. Retrieve a Gene Sequence form NCBI, and Determine the Exact Location
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lecture 17. Compare Different mRNA Transcripts and Select One to Evaluate a Gene
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Lecture 18. Understand Primer3 Setting + Primer Self-complementrity score
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Lecture 19. Calculate Primer Self-Complementarity Score
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Lecture 20. Check for Primer Cross Homology Using BLAT
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Lecture 21. Evaluate Primers Depending on Delta G
