Isolation Of Dna

Question 1

What is step one of isolation of dna and what three mechanisms

Answer 1

Cell lysis
Biologically
Physically
Or mechanically

Question 2

What are the biological options of cell lysis

Answer 2

Cellulase enzymes for plant membranes

Lysozymes for bacterial peptidoglycan wall

Sappanin for eukaryotic membrane breakdown

(All detergents)

Question 3

What are the 2 physical methods of cell lysis

Answer 3

Using osmolarity. Place cell in a hypotonic solution so water moves in

Or

Freeze thaw. Freezing will cause ice Crystal formation breaking cell membrane

Question 4

What are the 4 ways to mechanically lyse cells

Answer 4

Bead mill (beats and grinds sample)
Vortex
Pestle and mortar
Shearing

Question 5

What are the 3 shearing techniques to mechanical lysis

Answer 5

Homogenisers- squeeze fell through small space

Rotor stator - turbulence and mechanical shearing

Syringe needle- for long dna fragments

Question 6

What is step 2 of isolation

Answer 6

Dna purification/extraction

Question 7

What 2 ways are used to purify/extract dna from lysed cells

Answer 7

Phenol chloroform extraction

Or

Commercial kits (silica membrane)

Question 8

Explain phenol chloroform extraction of dna

Answer 8

Lysed cells mixed with phenol chloroform
This doesn’t mix with water. Aqueous layer forms on top with dna

Proteins stay in middle

Phenol chloroform sinks to the bottom

Dna carefully extracted from the aqueous layer

Question 9

Why is dna extraction using phenol chloroform bad

Answer 9

Chloroform is a anaesthetic

Phenol causes chemical burns

Question 10

What is an advantage of phenol chloroform extraction

Answer 10

Cost effective and fast

Question 11

How is dna purified from salts and debris after phenol chloroform extraction

Answer 11

Alcohol used to concentrate the dna removing salts

Question 12

How are commercial kits with silica membranes used to extract dna

Answer 12

Column allows dna to stick when high salt buffer is added

Salts and debris are washed away

Elution low salt buffer added to remove dna

Question 13

Why is commercial kits better than phenol chloroform

Answer 13

Less hazardous and more pure

Question 14

What is an issue with commercial kits

Answer 14

Expensive than phenol chloroform
And
Only small amount of dna sticks to silica

Question 15

How would you measure purity and quantity of dna

Answer 15

Spectrophotometry or nano drop using uv absorbance to detect levels

Gel electrophoresis

Capillary electrophoresis

Sybr green/ fluorescence

Question 16

What value on spectrophotometer shows dna is pure

Answer 16

1.8

Question 17

What 2 things are restriction endonucleases used for

Answer 17

Cloning

Cut dna for fingerprinting or for rflp PCR detecting snps

Question 18

Why do bacteria naturally have REa

Answer 18

Viral dna defence mechanism

Question 19

Where do RE cleave ds

Answer 19

Both 3’oh phosphodiester bonds between a dexoribose and phosphate

Question 20

Why is buffer added with mg to REa

Answer 20

Mg needed to cut bond

Also need right ph etc

Question 21

What is RE cutting at wrong sites due to contamination/chemicals or wrong buffer called

Answer 21

Star activity

Question 22

Where does agarose come from

Answer 22

Seaweed

Question 23

Agarose has pores. Why would you increase concentration of agarose from tae buffer

Answer 23

To increase these small pores to seperate smallest dna fragments