Primer Design For Polymerase Chain Reaction-Tips & Tricks
5.0 (3 ratings)
Instead of using a simple lifetime average, Udemy calculates a course's star rating by considering a number of different factors such as the number of ratings, the age of ratings, and the likelihood of fraudulent ratings.
33 students enrolled
Wishlisted Wishlist

Please confirm that you want to add Primer Design For Polymerase Chain Reaction-Tips & Tricks to your Wishlist.

Add to Wishlist

Primer Design For Polymerase Chain Reaction-Tips & Tricks

DNA primer design, what your mentor never taught you
5.0 (3 ratings)
Instead of using a simple lifetime average, Udemy calculates a course's star rating by considering a number of different factors such as the number of ratings, the age of ratings, and the likelihood of fraudulent ratings.
33 students enrolled
Created by Sarierah Jamil
Last updated 8/2016
English
Price: $20
30-Day Money-Back Guarantee
Includes:
  • 1 hour on-demand video
  • 5 Articles
  • 1 Supplemental Resource
  • Full lifetime access
  • Access on mobile and TV
What Will I Learn?
  • 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.
View Curriculum
Requirements
  • Interest in learning
  • Open mind
Description

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. 

Who is the target audience?
  • Biology students can take this course
Students Who Viewed This Course Also Viewed
Curriculum For This Course
24 Lectures
01:13:34
+
Introduction
1 Lecture 02:14
+
Polymerase Chain Reaction
3 Lectures 09:16

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.
Preview 03:06

This video explains in details:

  • The standard PCR amplification program.
  • The exponential amplification.
Lecture 2. Standard Amplification Program + Exponential Amplification
03:13

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!

Lecture 3. The size difference between the PCR amplification products of the 1st
02:57

Quiz
10 questions
+
PCR Primer Design Criteria
12 Lectures 36:24

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.
Lecture 4. List of the PCR Primer Design Criteria + Two Resources
01:44

How does the primer length play a role in the designing criteria?

Well, let's find out in this lecture.

Lecture 5. The DNA Primer Length
02:54

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.

Preview 03:56

Enthalpy, entropy, and free energy are somehow difficult for the beginners to understand instantly. This lecture explains them in a very easy way...

Lecture 7. How to Calculate Enthalpy, Entropy, and Free Energy
05:25

The nearest-neighbor method is more complicated than the other melting temperature calculation methods. In this lecture we simplified the complicated parts for you.

Lecture 8. Primer Tm-The Nearest Neighbor Method
02:54

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?
Lecture 9. Annealing Temperature (Ta) of the DNA Primer
03:02

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?
Lecture 10. GC-Content of the DNA Primer
02:30

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.
Lecture 11. GC Clamp at the 3’ End of the DNA Primer
04:01

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.
Lecture 12. Primer Secondary Structure
05:25

This lecture explains:

  • What do we mean by a primer with runs?
  • What do we mean by a primer with repeats?
Lecture 13. Runs & Repeats in the primer
02:54

Lecture 14. Cross homology
00:49

Learn how to measure the primer product length.

Lecture 15. Primer product length
00:50

Quiz- 2
9 questions

Quiz- 3
9 questions
+
Tools and Methods (How to...)
6 Lectures 23:15
Lecture 16. Retrieve a Gene Sequence form NCBI, and Determine the Exact Location
06:13


Lecture 18. Understand Primer3 Setting + Primer Self-complementrity score
04:39

Lecture 19. Calculate Primer Self-Complementarity Score
00:50

Lecture 20. Check for Primer Cross Homology Using BLAT
02:28

Lecture 21. Evaluate Primers Depending on Delta G
04:09
+
Gel Electrophoresis Troubleshooting
2 Lectures 03:23

Study multiple cases that results in the low intensity of DNA bands

Lecture 22. Low Intensity of All or Some DNA Bands
01:42

Study multiple cases that results in smeared DNA bands

Lecture 23. Smeared DNA Bands
01:41
About the Instructor
Sarierah Jamil
5.0 Average rating
3 Reviews
33 Students
1 Course
Easy Experiment Academy

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.