Mini Prep

Question 1

What is plasmid?

Answer 1

A double stranded, circular extra chromosomal DNA of bacterium

Question 2

What is plasmid used for?

Answer 2

In recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA

Question 3

What are the three steps of purifying plasmid DNA?

Answer 3

Growth of bacterial culture
Harvesting and lysis of bacteria
Purification of plasmid

Question 4

How is alkaline lysis used?

Answer 4

Breaks the cells open to collect cell components

Question 5

Outline the alkaline lysis method

Answer 5

Bacteria containing plasmid grown
Lyses with buffer containing SDS and NaOH
Through agitation, precipitation, centrifugation and the removal of supernatant, the plasmid is isolated

Question 6

What does the SDS do in solution II?

Answer 6

Denatures cell proteins

Cleaves the phospholipid bilayer of the membrane

Question 7

What does the NaOH do?

Answer 7

Disrupts hydrogen bonding between DNA bases which converts dsDNA to single stranded DNA
Also breaks down cell wall

Question 8

Describe the re-suspension of bacterial pellet in alkaline lysis

Answer 8

Resuspend pellet in solution containing tris, EDTA, glucose and RNase A - normally called solution I

Question 9

What are the roles of the components of solution I?

Answer 9

EDTA - chelates Mg+ and Ca2+ to stop DNAase damaged the plasmid. Also destabilises the cell wall

Tris HCl - buffer

Glucose - maintains osmotic pressure so cells don’t burst

RNAase A - degrade cellular RNA when cells are lysed

Question 10

Explain the neutralisation with solution III in alkaline lysis

Answer 10

Addition of potassium acetate (solution III) to decrease the alkalinity of the mixture
plasmid DNA re-natures to dsDNA here
This dissolves in solution whilst everything else becomes a white precipitant

Question 11

How can you separate precipitant from solution?

Answer 11

Centrifugation

Question 12

Outline the cleaning and concentration in alkaline lysis

Answer 12

Clean out salts, EDTA, residual proteins, debris and RNase

Ethanol precipitation

Question 13

What is LB broth?

Answer 13

Nutrient rich medium which is primarily used for growth of bacteria
Usually includes peptides; casein peptones; trace elements and minerals

Question 14

How can bacteria be selectively grown?

Answer 14

Introduction of an antibiotic

In this instance it was kanamycin which was specific to the plasmid

Question 15

What observation can be made with LB broth when bacteria successfully cultures?

Answer 15

Gets more cloudy as bacteria grows

Question 16

Explain what the following component of the lysis buffer is for: tris hcl

Answer 16

Act as a buffer to maintain a stable pH

Question 17

Explain what the following component of the lysis buffer is for: Sucrose

Answer 17

Create an oncotic pressure

Question 18

Explain what the following component of the lysis buffer is for: EDTA, EGTA

Answer 18

Chelate divalent ions to prevent unwanted reaction

Question 19

Explain what the following component of the lysis buffer is for: sodium fluoride and sodium pyrophosphatase

Answer 19

Protease inhibitors to prevent enzymatic breakdown of the protein

Question 20

Explain what the following component of the lysis buffer is for: Triton X

Answer 20

Detergent

Question 21

What are the components of the lysis buffer?

Answer 21

Tris HCl
Sucrose
EDTA
EGTA
Sodium fluoride 
Sodium pyrophosphatase
Triton X

Question 22

What are the advantages of using a lysis buffer?

Answer 22

Makes protein extraction simpler
Done without specialised equipment
Done at room temp on desk

Question 23

What are the disadvantages of using a lysis buffer at room temperature?

Answer 23

Room temp may cause degradation of the protein

Question 24

What does this method remove from the pellet and leave you with?

Answer 24

Removes protein and chromosomal DNA
then salts and small molecules at alcohol precipitation
Then leaves you with plasmid DNA

Question 25

Explain the roles of the components of solution II?

Answer 25

NaOH - disrupts H bonding in the dsDNA bases to convert to ssDNA

SDS - Detergent. Denatures proteins in cells and solubilises the membrane

Question 26

Explain the roles of the components of solution III

Answer 26

K acetate - decreases alkalinity to allow renature of DNA

Glacial - Ph balance

Water - dsDNA can dissolve in water but genomic DNA cant. Helps denatured proteins clump together.

Question 27

What can be used instead of isopropanol to washed the DNA pellet?

Answer 27

Phenylchloroform

Question 28

Why is isopropanol used > phenylchloroform?

Answer 28

Nasty burns