LAB 4

Question 1

Genetic material

Answer 1

DNA

Question 2

Blueprint of life

Answer 2

DNA

Question 3

Step 1-5 in isolating DNA (memorize mo dapat to)

Answer 3

1. Tissue homogenization and cell lysis.
2. Denaturation and Separation of biomolecules
3. Precipitation of nucleic acids
4. Washing
5. Resuspension

Question 4

This step allows the disruption of the cell
membrane and the release of different
macromolecules (including nucleic
acids) from the cell. This can be
performed using mechanical, chemical,
and/or enzymatic methods.

Answer 4

Tissue homogenization and cell lysis

Question 5

The homogenate/lysate contains all biomolecules from the cell. It is necessary to separate the nucleic acids from the other biomolecules and cellular components. This step usually involves the use of detergents and chaotropic agents to denature proteins, followed by centrifugation.

Answer 5

Denaturation and separation of biomolecules

Question 6

The chemical nature of nucleic acid allows it to be separated from other biomolecules through precipitation. Commonly, nucleic acid precipitation makes use of monovalent cations, alcohols, or both, to precipitate nucleic acids from a solution.

Answer 6

Precipitation of nucleic acids

Question 7

It is necessary to wash the nucleic acid pellet with 70% ethanol to remove the remaining salts, denaturants, and other contaminants. This is done to increase the purity of the DNA isolate.

Answer 7

Washing

Question 8

The DNA pellet can be resuspended in molecular grade water or Tris- EDTA solution prior to experimental use.

Answer 8

Resuspension

Question 9

the DNA must be subjected to quality assessment and quantification.

Answer 9

Post isolation

Question 10

Determination of the quality and quantity can be accomplished in two ways

Answer 10

gel electrophoresis
UV spectrophotometry

Question 11

DNA can be diluted and run on an
_____ to determine its quantity and quality

Answer 11

Agarose gel

Question 12

DNA fragments, including molecular
weight ladders are often heated to ____ prior to electrophoresis to straighten any loops formed along the length of the molecules so that migration through the gel is uniform

Answer 12

65 C

Question 13

The gel is exposed to _____, a flat molecule that intercalates, or slides between, the stacked base pairs of the DNA

Answer 13

Ethidium bromide

Question 14

Ethidium bromide fluoresces ____ in UV
light, making it possible to visualize the DNA

Answer 14

orange

Question 15

TRUE OR FALSE: If the DNA is intact, it will appear as a distinct band on the gel.

Answer 15

TRUE

Question 16

TRUE OR FALSE: If it is degraded, it will appear as a smear of thousands of small fragments.

Answer 16

True

Question 17

If the DNA is contaminated with protein, there will be a _______ of DNA at the bottom of the well and along the migration path from the wells where the slower moving protein trapped DNA

Answer 17

bright band

Question 18

If the well is overloaded with DNA that is too concentrated, the band will have a _____ smear above it.

Answer 18

Jagged

Question 19

RNA, on the other hand, appear as two distinct bands smaller than the genomic DNA correspond to the

Answer 19

28S and 18S RNA fractions

Question 20

The second method of determining the
quality and concentration of DNA is by

Answer 20

UV Spectrophotometry

Question 21

laboratory instrument that measures the
intensity of light passing through a solution and compares this to the amount of light entering the solution.

Answer 21

Spectrophotometer

Question 22

Emits light of specific wavelengths
on a sample tube

Answer 22

Spectrometer

Question 23

measures the light transmitted through the tube.

Answer 23

Photometer

Question 24

It includes a ______ cell that is
sensitive to the emitted wavelengths of light

Answer 24

Photoelectric cell

Question 25

A _____ that records the electrical potentials from the photoelectric cell.

Answer 25

Galvanometer

Question 26

The absorbance of UV light at a wavelength of _____ is used to determine double- stranded DNA concentration.

Answer 26

260nm

Question 27

The equation for calculation of DNA concentration is given by:

Answer 27

A260 x 50 ug/mL x dilution factor

Question 28

Protein in a sample can be detected at

Answer 28

A280

Question 30

The relative amount of protein contamination for a DNA sample can be calculated by

Answer 30

A260/A280 ratio

Question 31

Highly purified ratio

Answer 31

1.8 to 2.0

Question 32

Higher 2.0

Answer 32

DNA is contaminated with chloroform/phenol

Question 33

The absorbance at ____ nm is also used in assessing the quality of the DNA isolate

Answer 33

230 nm

Question 34

the A260 /A230 ratio should fall between

Answer 34

1.5 to 1.8

Question 35

Since both DNA and RNA absorb at Az60, if there is ____ in the sample, the calculated concentration of double stranded DNA will be inaccurately high.

Answer 35

RNA

Question 36

Running a sample on an ______ separates DNA from RNA and can be used to determine if RNA is present

Answer 36

Agarose gel

Question 37

What is Beer-Lamber's equation

Answer 37

n: A = 3cl where A = UV absorbance, & = wavelength dependent extinction coefficient, c = nucleic acid concentration and I = light path (cm).

Question 38

Gold standard for DNA quatification

Answer 38

260nm

Question 39

T presence of contaminants such as salts, proteins, and nucleotides. In addition, very low concentration of DNA (<_ g/mL) cannot be quantified using absorbance at 260nm.

Answer 39

2

Question 40

These dyes fluoresce when bound to dsDNA. The fluorescence is detected by a

Answer 40

Spectrofluorometer

Question 41

To quantify the DNA concentration, a standard curve is prepared using ____ or _____ in concentrations from 1 ng/mL to 1000 ng/mL. Depending on the standards used, this method can detect DNA as low as 25 pg/mL.

Answer 41

bacteriophage A or Calf thymus DNA

Question 42

It acts as a buffering agent to maintain a stable pH during DNA extraction, protecting DNA from degradation.

Answer 42

Tris Hydrochloride Acid

Question 43

It chelates divalent metal ions like Mg²⁺, inhibiting DNase activity and preventing DNA degradation.

Answer 43

Ethylenediaminetetraacetica acid

Question 44

It helps in stabilizing the DNA structure and promotes the precipitation of DNA by neutralizing its negative charge.

Answer 44

Sodium Chloride

Question 45

It is a detergent that lyses cell membranes and binds to polysaccharides, helping to purify DNA.

Answer 45

Cetyltrimethylammonium bromide

Question 46

This mixture separates proteins and other contaminants from DNA during phase separation.

Answer 46

Chloroform:Isoamyl Alcohol

Question 47

It precipitates DNA by reducing its solubility, allowing it to form a visible pellet.

Answer 47

Isopropanol

Question 48

It washes the DNA pellet to remove salts and other impurities while preserving the DNA structure.

Answer 48

70% Ethanol

Question 49

Separation of DNA or RNA from the other component of the cell

Answer 49

Nucleic Acid Isolation

Question 50

Tissue Homogenization or lysis. Mechanical method

Answer 50

Sonication and Homogenizer

Question 51

involves the use of ultrasonic waves to generate localized areas or high pressure resulting in cavitation that can shear apart cells

Answer 51

Sonication

Question 52

Chemical method of tissue homogenization or lysis

Answer 52

1. Buffer (Tris – HCL) ii. Salt (NaCl) iii. Cell lysis reagents (SDS) iv. Denaturants (Guanidine isothiocyanate) v. Reducing agents (beta- mercaptoethanol)

Question 53

Enzymatic method of tissue homogenization or lysis

Answer 53

Cellulase and Pectinase

Question 54

Denaturation and Separation of other cell biomolecules from the nucleic acid Chemical Treatment

Answer 54

i Phenol: chloroform ii. SDS iii. Protein denaturants

Question 55

Denaturation and Separation of other cell biomolecules from the nucleic acid Enzymatic

Answer 55

i. Protease ii. Proteinase

Question 56

Precipitation of Nucleic Acid a. Monovalent Cations

Answer 56

Sodium, Potassium, Ammonium

Question 57

Precipitation of Nucleic Acid a. Alcohols

Answer 57

i. Ethanol (95% - absolute) ii. Isopropanol ii. Lithium chloride iv. Centrifugation

Question 58

Washing of DNA pellet uses

Answer 58

a. 70 – 80% ethanol b. Centrifugation

Question 59

Drying of Pellet and Dissolution of dried Pellet methods

Answer 59

a. Air Drying b. Dissolution is sterilized molecular grade water or Tris- EDTA c. Room temperature or at 50 – 55C water bath

Question 60

Post-Processing methods

Answer 60

a. RNase treatment if DNA Isolation b. DNase treatment if RNA isolation

Question 61

Quality checking and Quantification method

Answer 61

a. Gel Electrophoresis b. Spectrophotometric analysis c. Fluruometer

Question 62

Technique to physically sort out nucleic acid fragments primarily on the basis of molecular size

Answer 62

Gel electrophoresis

Question 63

is added to running buffer during the separation of DNA fragments by agarose gel electrophoresis. This binds to DNA by intercalating between base pairs which causes the DNA helix to partially unwind

Answer 63

Ethidium Bromide (EtBr)

Question 64

EtBr is deadly so other options is used

Answer 64

GelRed SYBR Safe MIDORI green XTRA

Question 65

Absorbance reading to assess NA isolate quality and quantity methods

Answer 65

a. UV-VIS spectrophotometer b. Absorbance reading at 260nm (wavelength absorb by the N bases)

Question 66

Quantification of NA concentration using fluorometer methods

Answer 66

a. Use fluorescent dyes that bind to dsDNA or ssDNA b. Fluorescence when bound to DNA c. Detected by fluorometer d. Interpolation of concentration NA e. Calculated using line equation

Question 67

(1.8 - 2.0) is

Answer 67

High purity

Question 68

< 1.8 is

Answer 68

Protein contamination

Question 69

>2.0 is

Answer 69

chloroform / phenol contamination