MOL BIO LAB L6 (Midterms- DNA & RNA Extraction)

Question 1

One of the most pivotal steps in molecular biology, being routinely used in many areas of the biological and medical sciences, as this procedure marks a starting point in any molecular diagnostic kit.

Answer 1

Nucleic acid extration

Question 2

Encompass extraction of both DNA and RNA but can be more broadly characterized into chemically driven or solid phase methods.

Answer 2

Nucleic Acid Extraction (NAE) methods

Question 3

These methods rely on biochemical properties of the cellular components to elicit the desired molecular separation and might exhibit preference or exclusivity in extracting DNA or RNA, depending on its intrinsic characteristics.

Answer 3

Chemical methods of NAE

Question 4

Enumerate the four techniques under the chemical method of NAE

Answer 4

1. Osmotic shock
2. Enzymatic digestion
3. Detergents
4. Alkali treatment

Question 5

The only chemical technique that the mode of lysis is HARSH

Answer 5

Alkali treatment

Question 6

Principle of Osmotic Shock

Answer 6

Osmotic rupture of membrane

Question 7

The cost is moderate and is usually applied for general use

Answer 7

Detergents

Question 8

Principle of Enzymatic digestion

Answer 8

Digestion of cell wall

Question 9

Principle of Detergents and Alkali treatment

Answer 9

Solubilization of membrane

Question 10

Cheap at small scale; expensive at large scale

Answer 10

Enzymatic digestion

Question 11

Used for spheroplasts and protoplasts

Answer 11

Osmotic shock

Question 12

Used for gram-positive and gram-negative bacteria

Answer 12

Enzymatic digestion

Question 13

The cost of these chemical techniques is CHEAP

Answer 13

Osmotic shock and alkali treatment

Question 14

Used to extract DNA from bacteria and is based on the phenomenon of buoyant and specific density

Answer 14

Cesium Chloride (CsCl) Gradient Centrifugation with Ethidium Bromide (EtBr)

Question 15

Principle of Ultrasonication or cavitation

Answer 15

Disruption of cells by pressure

Question 15

Principle of Pressure cell (“French press”)

Answer 15

Disruption of cells by shear force

Question 15

The separation of RNA from DNA and proteins after extraction with an acidic solution, which consists mainly of GUSCN, sodium acetate, phenol, and chloroform, followed by centrifugation.

Answer 15

Guanidinium Thiocyanate-Phenol-Chloroform Extraction

Question 16

Enumerate the techniques under the Mechanical methods of NAE

Answer 16

1. Homogenization (blade or pestle)
2. Ultrasonication or cavitation
3. Pressure cell (“French press”)
4. Ball mill

Question 17

Principle of Homogenization (blade or pestle)

Answer 17

Shredding of cells

Question 18

Principle of Ball mill

Answer 18

Cells crushed between glass/steel/balls/beads

Question 19

The only mechanical technique that has a MODERATE mode of lysis

Answer 19

Homogenization (blade or pestle)

Question 20

The only mechanical technique that costs CHEAP

Answer 20

Ball mill

Question 21

A mechanical technique that costs moderate (method of choice for large scale)

Answer 21

Homogenization (blade or pestle)

Question 22

A mechanical technique that costs moderate to expensive

Answer 22

Ultrasonication or cavitation

Question 23

A mechanical technique that is used for animal tissues

Answer 23

Homogenization (blade or pestle)

Question 24

To evaluate DNA purity, measure absorbance from_______ to _______ to detect other possible contaminants.

Answer 24

230nm; 320nm

Question 24

A **mechanical technique** that is used for **Gram- negative** and some and **Gram positive** bacteria

Answer 24

Pressure cell (“French press”)

Question 25

A mechanical technique that is **good for** **spheroplasts** *but not primary cells*

Answer 25

Ultrasonication or cavitation

Question 25

A mechanical technique that used for bacteria, yeast, microalgae, unicellular animal cells

Answer 25

Ball mill

Question 26

The **most common purity calculation** is the ratio of the absorbance at?

Answer 26

260nm divided by the reading at 280nm. (260nm/280nm)

Question 26

The **most common technique** to determine DNA yield and purity is measurement of?

Answer 26

Absorbance

Question 27

What are needed for the absorbance method?

Answer 27

- Spectrophotometer equipped with a UV lamp - UV-transparent cuvettes (depending on the instrument) - Solution of purified DNA

Question 28

Where DNA **absorbs light most strongly**, and the number generated allows one to estimate the concentration of the solution.

Answer 28

260nm (A260)

Question 28

Absorbance readings are performed at?

Answer 28

260nm (A260)

Question 29

**Good-quality** DNA will have an A260/A280 ratio of?

Answer 29

1.7-2.0

Question 30

A reading of ____ **does not render the DNA unsuitable** for any application, but Iower ratios indicate more contaminants are present.

Answer 30

1.6

Question 30

DNA purity formula

Answer 30

A260/A280

Question 31

Formula used for correcting for turbidity

Answer 31

DNA purity (A260/A280)= (A260 reading — A320 reading) / (A280 reading — A320 reading)

Question 32

Can indicate that **organic compounds or chaotropic salts are present** in the purified DNA.

Answer 32

Strong absorbance around 230nm

Question 33

This ratio can **help evaluate the level of salt carryover** in the purified DNA.

Answer 33

ratio of 260nm to 230nm

Question 34

In terms of absorbance, the _____the ratio, the _____ the amount of thiocyanate salt is present.

Answer 34

lower; greater

Question 35

The A260/A230 is best if greater than?

Answer 35

1.5

Question 36

A reading at ____ will indicate **if there is turbidity** in the solution, another **indication of possible contamination**.

Answer 36

320nm

Question 37

**One optical density unit** (or absorbance unit) at **260 nm** is equivalent to?

Answer 37

50 mg/L (or 50ug/mL) of double-stranded DNA and 40 ug/mL of RNA.

Question 38

Formula for ***DNA* Concentration (ug/mL)**

Answer 38

(A260 reading — A320 reading) x dilution factor x ***50ug/ml***

Question 39

NOTE: *DNA concentration* is estimated by **measuring** the **absorbance at 260nm**, adjusting the A260 measurement for turbidity (measured by absorbance at 320nm), **multiplying** by the **dilution factor**, and using the relationship that an A260 of 1.0 = 50ug/ml pure dsDNA.

Question 40

Formula for ***RNA* Concentration (ug/mL)**

Answer 40

(A260 reading — A320 reading) x dilution factor x ***40ug/ml***

Question 41

DNA yield (ug) formula

Answer 41

DNA concentration x total sample volume (ml)

Question 42

This is the **limit for suitability** in terms of purity

Answer 42

2.5

Question 43

In Guanidinium Thiocyanate-Phenol-Chloroform extraction, this can be ***recovered*** **through precipitation by isopropanol** and can be used for subsequent process.

Answer 43

Total RNA

Question 44

In Guanidinium Thiocyanate-Phenol-Chloroform Extraction, ______ remains in the **upper aqueous phase**, while most of ______ and proteins part **remain either in the interphase** or in the **lower organic phase** under *acidic condition.*

Answer 44

Total RNA; DNA

Question 44

Dedicated to **plasmid DNA isolation**

Answer 44

Alkaline Extraction

Question 44

Used in the **field of forensics for DNA extraction** from various sources, such as **hair, blood stain cards, and buccal swabs**

Answer 44

Chelex® Extraction

Question 44

**Inhibits DNA degradation** by ***chelating metal ions*** which cause DNA breakdown at high temperature and lower ionic conditions

Answer 44

Chelex® Extraction

Question 45

What is the basic principle of Alkaline Extraction?

Answer 45

**selective alkaline denaturation** of high molecular weight chromosomal DNA, while **covalently bond circular plasmid DNA** remains intact.

Question 46

**Alkaline Extraction** involves harvesting the bacteria of interest from culture media and exposing them to alkaline solution (consisting basically of ______and ______ ).

Answer 46

SDS or sodium dodecyl sulfate; NaOH (sodium hyroxide)

Question 47

**Poly(A) RNA** ***hybridizes*** with an **oligo(dT)-cellulose matrix**, under high-salt conditions

Answer 47

Purification of Poly(A)+ RNA by Oligo(dT)-Cellulose Chromatography

Question 48

Based on **liquid and stationary phases,** which selectively separate the target analyte from the solution **based on specific hydrophobic, polar, and/or ionic properties** of both solute and sorbent.

Answer 48

Solid-Phase Nucleic Acid Extraction

Question 49

**Types(?)** of Solid-Phase Nucleic Acid Extraction

Answer 49

- normal/regular SPE - reverse SPE - ion exchange SPE

Question 50

Enumerate the **solid phases** of Solid-Phase Nucleic Acid Extraction use:

Answer 50

- silica matrices - Glass Particles - Diatomaceous Earth - Magnetic Beads-Based Nucleic Acid Purification (most common) - Anion Exchange Material - Cellulose Matrix