Tag: PCR

What are some examples of situations where a doctor might order a lab test to aid in the diagnosis and treatment of a patient?Why does the government regulate clinical laboratories? Why is CLIA certification required?

Lab Extra Credit Case Study

Questions

What types of samples might be collected from patients to use for laboratory testing?

What are some examples of situations where a doctor might order a lab test to aid in the diagnosis and treatment
of a patient?Why does the government regulate clinical laboratories? Why is CLIA certification required?

How does the work of a clinical laboratory differ from the experiments being conducted in a research laboratory?

Why don’t research laboratories have to comply with CLIA regulations?How does COVID-19 testing work? How do healthcare professionals make a diagnosis of COVID-19 infection? Let’s follow Marcus through the process.What samples are typically collected from patients suspected of coronavirus exposure?

Name three sources of nucleic acids you expect to find
in a patient sample.Compare and contrast traditional PCR with quantitative PCR.

How can we ensure we are only amplifying and detecting viral sequences, and no other sequences that may be in the tube?

Discuss these with each other or your supervisor-Analyse your plasmid DNA using agarose gel electrophoresis,

Genetics

BI2S109 Human Molecular Genetics

From gene to protein: overexpressing insulin and green fluorescent protein

Objectives
In these practicals you will learn how to:

• Set up a PCR and amplify a human gene,

• Set up a restriction digest,

• Ligate an insert into a plasmid vector and to transform bacteria,

• Use antibiotics to screen for transformants,

• Regulate the expression of the INS or GFP gene using arabinose,

• Calculate transformation efficiency,

• Isolate a plasmid,

• Analyse your plasmid DNA using agarose gel electrophoresis,

Overview
Prac 1
• Set up and perform PCR

• Digest plasmid with NheI to open plasmid between Ara promoter and GFP

• Check digest on gel
Prac 2
• Cut PCR fragment with NheI

• Ligate PCR product in plasmid

• Transform E. coli with plasmid on Ara/Amp plates

• Select glowing/non-glowing colonies (not actually happening in the prac)
Prac 3 (3hrs)
• Plasmid DNA extraction

• Restriction digest of plasmid

• Check digest on agarose gel

• Pick colony and plate bacterial cells on plates with and without arabinose

Will be completed for you and results made available to you on Blackboard:

• Check GFP production by glow

• Check GFP or Insulin protein production by protein SDS PAGE gel electrophoresis

• Transfer proteins to Western blot membrane

• Western blot detection

You need to record all the work in your lab book which you can use to write the lab report.

The questions in this manual are there to help to understand the practical.

Discuss these with each other or your supervisor.

Introduction
In this lab you will perform