Molecular Cell Biology Flashcards
Week 5 - three lectures
Recombinant DNA
Study proteins and produce in quantities
Techniques - produce (Clone), separate (SDS-PAGE), identify (Western Blot)
Plasmid Vector
Small pieces of DNA
Express gene in cells (circle shaped)
Antibody resistant (allows separation)
Polymerase Chain Reaction (PCR)
Amplify gene, insert into desired plasmid, insert cloned plasmid into bacteria cell
Bacteria Transformation in PCR
Bacteria replicates plasmids making it easy to purify large DNA quantities
Promoter in PCR
Drives gene expression in specific cells
Gene of interest PCR
Encodes protein
Antibiotic Resistance Gene PCR
Allows for selection of bacteria containing plasmid
PCR Workflow of DNA
Denature - separate strand (hot)
Anneal - bind to primers (cold)
Extension - polymerase to new strand (warm)
Cloning Process PCR
Linearize plasmid - open plasmid using PCR
Insert gene - user primer
Ligation - ligase to seal gene into plasmid
Transformation - plasmid into bacteria via thermal shock
Selection - grow bacteria on antibiotic plates
Purify - extract plasmid DNA from bacterial culture
Polymerase PCR
Synthesize original DNA to new strand
Linearize Plasmid - 1 cloning
Open circular plasmid using PCR
Primers - 2 cloning
Incorporate gene into plasmid, creates sticky ends
Ligation - 3 cloning
Use DNA ligase to seal gene in plasmid
Transformation - 4 cloning
Introduce plasmid into bacteria via electric shock
Selection - 5 cloning
Grow bacteria on antibodic plates to isolate those containing the plasmid
Purification - 6 cloning
Extract plasmid DNA from bacteria cultures
Sequencing
Confirm gene insertion and check for mutations during PCR
SDS-PAGE
Separate proteins based on molecular weight using electric field
Stacking & Resolving Gel - SDS-PAGE
Stack - lower pH and acrylamide concentration to force proteins into tight bands
Resolve - higher acrylamide to separate proteins by size during electrophoresis
Electrophoresis
Electric Field - applied to gel causing negative charged proteins to migrate towards positive electrode
Migration - smaller proteins move faster through gel mesh (allows size-based separation)
Coomassie Blue
Stain that binds to protein
Bands compared to molecular weight marker (ladder) to determine size
Western Blot
Detects protein from mixture using antibodies
Electric current moves protein from gel onto membrane “sandwich”
Western Blot - Workflow
Protein transfer - Electrophoresis (sandwich)
Blocking - cover areas without proteins (milk)
Antibody Incubation - primary / secondary
Detection - enzyme reaction (HRP catalyzes reaction) / signal detection (light emission)
