Molecular Cell Biology

Question 1

Recombinant DNA

Answer 1

Study proteins and produce in quantities
Techniques - produce (Clone), separate (SDS-PAGE), identify (Western Blot)

Question 2

Plasmid Vector

Answer 2

Small pieces of DNA
Express gene in cells (circle shaped)
Antibody resistant (allows separation)

Question 3

Polymerase Chain Reaction (PCR)

Answer 3

Amplify gene, insert into desired plasmid, insert cloned plasmid into bacteria cell

Question 4

Bacteria Transformation in PCR

Answer 4

Bacteria replicates plasmids making it easy to purify large DNA quantities

Question 5

Promoter in PCR

Answer 5

Drives gene expression in specific cells

Question 6

Gene of interest PCR

Answer 6

Encodes protein

Question 7

Antibiotic Resistance Gene PCR

Answer 7

Allows for selection of bacteria containing plasmid

Question 8

PCR Workflow of DNA

Answer 8

Denature - separate strand (hot)
Anneal - bind to primers (cold)
Extension - polymerase to new strand (warm)

Question 9

Cloning Process PCR

Answer 9

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

Question 9

Polymerase PCR

Answer 9

Synthesize original DNA to new strand

Question 10

Linearize Plasmid - 1 cloning

Answer 10

Open circular plasmid using PCR

Question 11

Primers - 2 cloning

Answer 11

Incorporate gene into plasmid, creates sticky ends

Question 12

Ligation - 3 cloning

Answer 12

Use DNA ligase to seal gene in plasmid

Question 13

Transformation - 4 cloning

Answer 13

Introduce plasmid into bacteria via electric shock

Question 14

Selection - 5 cloning

Answer 14

Grow bacteria on antibodic plates to isolate those containing the plasmid

Question 15

Purification - 6 cloning

Answer 15

Extract plasmid DNA from bacteria cultures

Question 16

Sequencing

Answer 16

Confirm gene insertion and check for mutations during PCR

Question 17

SDS-PAGE

Answer 17

Separate proteins based on molecular weight using electric field

Question 18

Stacking & Resolving Gel - SDS-PAGE

Answer 18

Stack - lower pH and acrylamide concentration to force proteins into tight bands
Resolve - higher acrylamide to separate proteins by size during electrophoresis

Question 19

Electrophoresis

Answer 19

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)

Question 20

Coomassie Blue

Answer 20

Stain that binds to protein
Bands compared to molecular weight marker (ladder) to determine size

Question 21

Western Blot

Answer 21

Detects protein from mixture using antibodies
Electric current moves protein from gel onto membrane “sandwich”

Question 22

Western Blot - Workflow

Answer 22

Protein transfer - Electrophoresis (sandwich)
Blocking - cover areas without proteins (milk)
Antibody Incubation - primary / secondary
Detection - enzyme reaction (HRP catalyzes reaction) / signal detection (light emission)