LESSON 6 LAB: PC POST PROCESSES, TYPES OF PCR AND PCR REAGENTS

What is DNA purification?

The process of removing PCR reagents, salts, enzymes, primers, and other contaminants to obtain purified DNA.

Why is DNA purification important?

It ensures clean DNA for downstream applications such as sequencing and cloning.

What is DNA sequencing?

A method for determining the exact order of nucleotide bases in a DNA molecule.

Why is DNA sequencing done?

To understand gene function and genome structure.

What is electrophoresis?

A method that separates DNA, RNA, or proteins based on size, charge, and binding affinity using a gel or fluid.

What is restriction digestion?

A procedure where restriction enzymes cut DNA at specific recognition sites to produce DNA fragments.

What are restriction enzymes?

Enzymes that recognize and cut DNA at specific sequences.

What is PCR cloning?

The amplification of DNA fragments and vectors using PCR followed by recombination into a vector.

What is a recombinant cloning vector?

A vector containing an inserted DNA fragment produced during cloning.

What does conventional PCR do?

Amplifies DNA using thermocycling and analyzes products with gel electrophoresis.

Why is conventional PCR considered baseline?

It detects DNA only at the end of PCR and requires post-PCR analysis.

What is real-time PCR (qPCR)?

A PCR method that monitors DNA amplification in real time using fluorescent markers.

What is the advantage of qPCR?

It is quantitative, faster, more sensitive, and does not require gels.

What is RT-PCR?

A PCR technique that converts RNA to cDNA before amplification.

Why is RT-PCR used?

For detecting RNA viruses such as COVID-19.

What is nested PCR?

A two-round PCR using two sets of primers for higher specificity.

Why use nested PCR?

It reduces nonspecific amplification and increases sensitivity.

What is multiplex PCR?

A PCR method that amplifies multiple targets in one reaction using several primer sets.

Why use multiplex PCR?

It saves time and sample but requires careful primer design.

What is touchdown PCR?

A method where annealing temperature starts high then decreases.

Why use touchdown PCR?

It improves specificity by minimizing nonspecific binding.

What is hot-start PCR?

A technique where polymerase is activated only at high temperatures.

Why use hot-start PCR?

It prevents premature or nonspecific DNA amplification.

What is digital PCR (dPCR)?

A method dividing samples into thousands of reactions for absolute quantification.

What is the advantage of digital PCR?

Provides high precision without standard curves.

What is high-fidelity PCR?

PCR using polymerases with proofreading ability.

Why use high-fidelity PCR?

It produces fewer errors, ideal for cloning and sequencing.

What is long-range PCR?

PCR designed to amplify DNA fragments greater than 10 kb.

Why use long-range PCR?

To amplify large genomic regions that conventional PCR cannot.

What is the purpose of nuclease-free water in PCR?

To maintain reaction volume and prevent nuclease contamination.

What does 10X PCR buffer do?

Provides the correct chemical environment for enzyme activity.

What are dNTPs used for in PCR?

They are the nucleotide building blocks for new DNA strand synthesis.

What is the role of MgCl2 in PCR?

Provides Mg2

What does the forward primer do?

Defines where DNA synthesis begins on the target strand.

What does the reverse primer do?

Binds the opposite strand to define the other boundary of the target region.

What is the role of the DNA sample in PCR?

Serves as the template containing the sequence to be amplified.

What does Taq polymerase do?

Synthesizes new DNA strands and withstands high temperatures.

What are micropipettes used for?

To measure and transfer very small liquid volumes accurately.

What are pipette tips for?

To prevent contamination during liquid transfer.

What are PCR tubes used for?

To hold the reaction mixture during PCR thermal cycling.

What are Eppendorf tubes used for?

For preparing, mixing, and storing reagents before PCR setup.

What does a vortex mixer do?

Mixes reagents thoroughly for even distribution.

What does a mini-centrifuge do?

Brings liquids to the bottom of tubes to avoid reagent loss.

What is the thermal cycler used for?

Performs temperature cycling needed for DNA denaturation, annealing, and extension.

What is an Eppendorf tube rack for?

To hold tubes upright for stable and accurate pipetting.

Why wear nitrile gloves during PCR?

To prevent contamination and protect the user.

What is 70% alcohol used for in PCR setup?

To disinfect tools and surfaces.

What are tissues or wipes used for in PCR labs?

To clean spills and equipment surfaces.

What is the purpose of a bin for tips?

Provides safe disposal for contaminated pipette tips.