Build Strong Foundations- PCR, Multiplex PCR, DNA Sequencing

In this one of a kind course, students will gain understanding of PCR with efficient utilization achieved by conscious efforts ingrained by the learning to improve each step of the PCR process. In the end, the learner will have the advantages of time saving and cost saving when the need for multiplex PCR amplification arises.

Keeping this mind, the lectures are divided into sections-  Improving the moving parts of PCR which covers PCR and multiplex PCR. The  second learning section covers DNA sequencing.

The  best way to  organize the environment for PCR set up are discussed.

The lecture is an overview of PCR consumables in a nut shell at an introductory level.

DNA Polymerase role in PCR is explained.

Preparation calculations for stock and working primer from lyophilized primers.

Preparation calculations for stock primer from lyophilized primers (supplied in ug).

A Primer on Polymerase Chain Reaction primers  explains PCR primer characteristics and  mechanism.

The lecture touches upon the topic of type of PCR to choose depending on application.

Increase the efficiency of your PCR primers by following these simple, but often, overlooked steps.

The lecture discusses how  PCR primer design step be used for adding Nde I restriction enzyme site to PCR product.

Polymerase Chain Reaction describe the PCR exponential amplification, basic reaction setup from beginning to end.

Polymerase Chain Reaction thermal Profile focuses on  "turning heat" on DNA to behave in Polymerase Chain Reaction to achieve  efficient gene amplification. Concepts of melting temperature,denaturation temperature, annealing temperature and extension temperature in the context of gene amplification are discussed.

Describes common  Primer related PCR trouble shooting with solutions.

PCR additives lecture give an overview of use of additives to enhance PCR efficiency for difficult gene regions.

What is Cycle Sequencing PCR ?

Learners will gain  experience of preparing 2% agarose for electrophoresis and why agarose remains an attractive option for DNA/PCR product separation.

DNA staining used in routine molecular  biology for PCR products and native DNA are discussed.

Nothing is more disheartening to a molecular bench scientist or technician than continuous PCR failures with no solutions in sight. This lecture shows how to start your investigations to figure out the mistakes or reasons for the PCR failures.

This is  section 1 in concentrated form.

This lecture gives an overview of multiplex PCR and act as the bridge to connect to the lecture - Designing multiplex PCR.

The course multiplex PCR demonstrate how we could use it for efficient mutation screening with savings of time and reagents. The downloadable article (RPE65.pdf) shows the adoption of multiplex PCR for Retinal Pigment Epithelium Gene 65 mutation screening.

The lecture describes use cases of multiplex PCR in specific scenarios.

This summary lecture gives demonstration of  PCR workflow from downloading gene sequences to PCR set up and electrophoresis.

Learn an overview of sanger sequencing method.

Fluorescence-based detection enhanced fast adoption of  automation for  single gene sequencing and also  resulted in routine use of complete exon by exon gene scanning for mutations. This  led to increased reliability of gene mutation study results with utility in clinical diagnosis and management. In this lecture, sequencing principle and basic gene sequencing protocol is discussed.

Analysis of gene sequence output(from raw data to meaningful parameters) is the critical step in mutation detection by all methods including Next Generation Sequencing methods. In "Sequencing Data Analysis for the beginner", the basic sequence analysis technique sufficient enough to understand the principles are discussed.

The referenced DNA sequence softwares in this lecture will help you to familiarize with easy to use tools in practice in molecular biology.

The lecture on gene mutations covers mutations types with examples and supplemental reading materials.