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
