PCR - Cloning and PCR Techniques

Flashcards covering key concepts and details about cloning methods and PCR techniques as discussed in the lecture.

What are the four simple steps to cloning PCR products?

Template DNA, Denaturing dsDNA, Annealing of Primers, Synthesizing new DNA.

What does TOPO cloning rely on?

The enzyme DNA topoisomerase I, which functions as both an endonuclease and a ligase.

What characteristic of Taq polymerase contributes to its error rate?

Taq polymerase lacks 3’ to 5’ exonuclease proofreading activity.

What does adding restriction enzyme sites to 5’ ends of PCR primers facilitate?

Cloning into complementary sites in another vector.

What is the purpose of positive controls in PCR experiments?

To validate that the experimental system is functioning correctly by providing known positive results.

What is Nested PCR?

A type of PCR where specificity is improved using two sets of primers sequentially.

Why is Phusion® High-Fidelity DNA Polymerase preferred?

Because it has increased fidelity and speed due to its unique structure.

What is the outcome when using degenerate primers in PCR?

They cover all possible nucleotide combinations for a given protein sequence.

How does the pJET1.2 vector facilitate cloning?

It is designed for blunt-end cloning with restriction enzyme sites for later manipulation.

What is the role of negative controls in PCR?

They validate positive results by ensuring that any amplification occurred only due to the presence of the target DNA.