Purification of nucleic acids 1

Question 1

Why does cloning have purifying properties

Answer 1

Gene cloned among mix of fragments. Only 1 recombinant molecule introduced into each cell, so each individual clone made only has copies of 1 recombinant DNA molecule

Question 2

Transformation

Answer 2

Introduction of plasmid DNA into a bacterial cell
cold Cacl2 followed by heat shock
inefficient

Question 3

Infection

Answer 3

infection of bacterial cell by complete bacteriophage
efficient
Phage nucleic acid in protein coat

Question 4

Transfection

Answer 4

Introduction of naked bacteriophage DNA into bacterial cell by transformation
Cold Cacl2 then heat shock

Question 5

Steps of nucleic acid purification

Answer 5

Growing medium, Cell lysis, cell extract treated to remove all components except DNA (phenol, ion exchange chromatography), concentration

Question 6

Growing bacteria

Answer 6

defined/undefined medium
late exponential phase
centrifuge at end- cell extract forms pellet

Question 7

Cell lysis

Answer 7

Pellet put in lysis solution, disrupts CW, CSM
Detergent removes lipids from CSM
Centrifuge, insoluble debris forms pellet
DNA, RNA and protein in solution

Question 8

Removing proteins and RNA

Answer 8

Add protease and phenol, centrifuge
phenol denatures proteins and protease digests them
Proteins left as white coagulated mass at interface, between aqueous and organic layer
Separate RNA and DNA by ion-exchange chromatography

Question 9

Concentration of DNA

Answer 9

Add ethanol and salt, form layers on top
If DNA at high concentration, remove using glass rode
If low, mix centrifuge to collect precipitate