Lesson 3 - Nucleic Acid Isolation

Question 1

application of nucleic acid extraction

Answer 1

1. DNA profiling
2. molecular biotechnology
3. phylogenetic studies

Question 2

where is high quality used for

Answer 2

1. restriction digestion
2. gene cloning
3. amplification
4. DNA sequencing

Question 3

Common sources of DNA

Answer 3

1. whole blood
2. hair
3. sperm
4. bones
5. nails
6. tissues
7. saliva

Question 4

What are the five general steps in nucleic acid isolation

Answer 4

1. tissue homogenization and cell lysis
2. denaturation and separation of other biomolecules from nucleic acid
3. precipitation of nucleic acid from aqueous phase
4. washing of precipitated nucleic acid
5. drying of pellet and dissolution of dried pellet

Question 5

mechanical method for tissue homogenization

Answer 5

1. sonication
2. grinding

Question 6

refers to the process of applying sound energy to agitate particles or discontinuous fibers in a liquid

Answer 6

Sonication

Question 7

reduce the size of materials to give a usable form or to separate their components

Answer 7

grinding

Question 8

chemical used for cell lysis

Answer 8

1. buffer
2. salt
3. detergent/surfactant
4. denaturants

Question 9

buffer

Answer 9

Tris-HCl

Question 10

salt

Answer 10

NaCl

Question 11

detergent/surfactant

Answer 11

SDS (sodium dodecyl sulfate)

Question 12

purpose of detergent

Answer 12

emulsify phospholipids

Question 13

denaturants

Answer 13

guanidinium

Question 14

what does guanidinium do

Answer 14

inactivates RNases / DNases

Question 15

used to treat other cellular components

Answer 15

enzymatic treatment

Question 16

enzymatic treatment examples

Answer 16

• lysozyme
• cellulase
• pectinase

Question 17

chemical treatment for denaturation and separation of other biomolecules from the nucleic acid

Answer 17

1. phenol
2. choloroform
3. isoamyl alcohol
4. CTAB
5. PVP

Question 18

denature proteins

Answer 18

phenol

Question 19

what does phenol denature

Answer 19

proteins

Question 20

removes proteins and lipids

Answer 20

chloroform

Question 21

what does chloroform remove

Answer 21

proteins and lipids

Question 22

removes phenol and chloroform

Answer 22

isoamyl alcohol

Question 23

what does isoamyl alcohol remove

Answer 23

phenol and chloroform

Question 24

removes polysaccharides

Answer 24

CTAB (cetyltrimethylammonium bromide)

Question 25

what does CTAB (cetyltrimethylammonium bromide) remove

Answer 25

polysaccharides

Question 26

removes polyphenols (alkaloids)

Answer 26

PVP (polyvinylpyrrolidone)

Question 27

what does PVP (polyvinylpyrrolidone) remove

Answer 27

polyphenols (alkaloids)

Question 28

used in enzymatic treatment during the denaturation and separation of other biomolecules from the nucleic acid

Answer 28

- protease - proteinase

Question 29

sample will be separated by density with heavier stuff at the bottom of the tube and lighter stuff on top

Answer 29

centrifugation

Question 30

formed during centrifugation

Answer 30

1. supernatant 2. precipitate

Question 31

clear liquid free of precipitate located above the solid part

Answer 31

supernatant

Question 32

solid that forms at the bottom after centrifugation

Answer 32

precipitate

Question 33

monovalent cations

Answer 33

- sodium - potassium

Question 34

alcohol used in the precipitation of nucleic acid from aqueous phase

Answer 34

- ethanol (95%) - isopropanol (95%)

Question 35

used to wash precipitated nucleic acids

Answer 35

70% ethanol / isopropanol

Question 36

two types of dyring of pellet

Answer 36

1. air drying 2. vacuum drying

Question 37

where is dissolution of dried pellet done

Answer 37

- sterilized molecular grade water or - TE (Tris-EDTA)

Question 38

EDTA

Answer 38

Ethylenediaminetetraacetic acid

Question 39

inactivates DNases

Answer 39

EDTA

Question 40

what does EDTA inactivate

Answer 40

DNases

Question 41

after isolation, __ treatment if DNA is isolated

Answer 41

RNA

Question 42

after isolation, __ treatment if RNA is isolated

Answer 42

DNA

Question 43

storage of isolated DNA

Answer 43

1. stock solution 2. working solution

Question 44

stock solution temp.

Answer 44

-20°C to -80°C

Question 45

working solution temp.

Answer 45

4°C

Question 46

stock solution

Answer 46

pellet + Tris-EDTA

Question 47

how to determine purity and concentration of DNA isolation

Answer 47

1. using DNA standard (different concentrations) 2. UV spectrophotometer (purity)

Question 48

laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size.

Answer 48

Gel electrophoresis

Question 49

To assess purity using the UV spectrophotometer, what is done?

Answer 49

1. take readings at 260 nm - 280nm 2. get ratio of A260/A280 3. get ratio of A260/A230

Question 50

high purity for NA sample

Answer 50

A260/A280 = 1.8 to 2.0

Question 51

A260/A280 < 1.8 (or <1.6)

Answer 51

protein contamination

Question 52

protein contamination

Answer 52

A260/A280 < 1.8 (or <1.6)

Question 53

A260/A280 > 2.0

Answer 53

chloroform/phenol contamination

Question 54

chloroform/phenol contamination

Answer 54

A260/A280 > 2.0

Question 55

if A260/A230 < 2.0, it is contaminated with:

Answer 55

1. carbohydrate carryover 2. residual phenol/guanidinium 3. other organic compounds 4. salts