Theory and Technique

Question 1

Why is it difficult to isolate pure nucleic acids from cells?

Answer 1

Because both DNA and RNA are usually found complexed with proteins within the cell

Question 2

Why is RNA hard to extract from cells?

Answer 2

Because it is bound up with proteins to make ribonucleoprotein complexes such as ribosomes

Question 3

What are three basic methods commonly used to deproteinize cellular extracts?

Answer 3

• Highly purified protease solutions that degrade protein
• Strong salts to dissolve nucleic acids and precipitate denatured protein
• Phenol can denature protein

Question 4

Where are ribonucleases found?

Answer 4

In most living cells and in breath and perspiration

Question 5

What are Sarkosyl and SDS used for when isolating DNA and RNA?

Answer 5

To denature ribonucleases that may degrade RNA

Question 6

What do nucleases require for activity? What can stop their activity do to this fact?

Answer 6

Most need magnesium ions (divalent cations), chelating agents such as EDTA are often used in nucleic acid extraction .

Question 7

Why are nucleic acid extractions carried out on ice?

Answer 7

To minimize nuclease activity

Question 8

What gives a phenol solution a yellow colour? What else does this additive do?

Answer 8

8-hydroxyquinoline

Chelating agent that soaks up divalent cations

Question 9

After a phenol extraction, what do the three layers represent?

Answer 9

• Nucleic acids (upper aqueous layer)
• Denatured protein (middle phenol-aqueous interface phase)
• Hydrophobic cell components (lower phenol phase)

Question 10

Phenol extractions are completed until?

Answer 10

No further protein is observed at the phenol-aqueous interface

Question 11

What concentration of ethanol precipitates nucleic acid and what concentration removes traces of phenol?

Answer 11

• Absolute (near 100%) ethanol precipitates nucleic acids

- 80% removes phenol from pellet

Question 12

How is high MW RNA precipitated?

Answer 12

Strong solutions of sodium chloride

Question 13

How is DNA precipitated?

Answer 13

10% w/v polyethylene glycol (PEG)

Question 14

Why is the presence of a simple salt (eg. sodium acetate) required with ethanol precipitation to separate nucleic acid fractions from PEG NaCl or phenol?

Answer 14

The facilitate the aggregation of charged nucleic acid molecuels

Question 15

In which order are nucleic acid fractions fractionated?

Answer 15

• High MW RNA
• DNA
• Low MW RNA

Question 16

What are the components of a nucleic acid extractino buffer?

Answer 16

• Tris-HCL
• EDTA-Na
• Sarkosyl
• Nacl

Question 17

Purines and pyrimidines absorb light strongly at ____ nm

Answer 17

260 nm

True for both RNA and DNA

Question 18

Proteins exhibit a strong absorption at ____ nm. Why?

Answer 18

280 nm

Aromatic amino acid content (tyrosine and tryptophan)

Question 19

Polysaccharides show strong absorbances around ___ nm

Answer 19

230

Question 20

For pure nucleic acids 260/280 = ?

Answer 20

1.8

Question 21

A nucleic acid sample with a 260/280 value less than 1.8 has what sort of impurities?

Answer 21

Protein

Question 22

A nucleic acid samples with a 260/280 value more than 1.8 has what sort of impurities?

Answer 22

Buffer contamination

Question 23

What absorbance wavelength is used to determine the quantitative estimation of the amount of nucleic acid present?

Answer 23

A260

Question 24

What is a specific extinction coefficient defined as?

Answer 24

The theoretical absorbance of a 1% solution at a given wavelength and light path length of 1 cm.

Question 25

The relationship between absorbance and concentration is based on what Law?

Answer 25

The Beer-Lambert law A = KcL ``` A = absorbance K = Constant c = concentration L = Length of light path (cm) ```

Question 26

What is the simplified formula derived from the Beer Lambert Law that allows the concentration of a nucleic acid sample to be determined?

Answer 26

[nucleic acid] = (A260)(concentration at A260=1 ug/mL)(dilution factor) Unit conversions are necessary

Question 27

What sort of spectrophotometric lamp conditions should be in place in a spectronic 601?

Answer 27

B (tungsten and deuterium) or D (deuterium only)

Question 28

What are the default concentration factors to be multiplied by A260 for DNA, RNA and oligonucleotides?

Answer 28

DNA: 50 RNA: 40 Oligonucleotides: 33

Question 29

Why should the absorbance of nucleic acids be greater than .1 at 260 nm?

Answer 29

For accurate measurements of 260/280 purity check

Question 30

Small variations at what wavelength affects the 260/280 value of nucleic acids more?

Answer 30

280 nm

Question 31

What are the two purposes for a running buffers solution during agarose gel electrophoresis?

Answer 31

- Keeps the gel cool as it runs | - Contains ions that will conduct the electricity

Question 32

What are three purposes for gel loading buffer?

Answer 32

- Increase density of the sample to insure that it drops into the well - Adds colour to the sample - Contains migratable dyes that move toward the anode at a predictable rate

Question 33

Ethidium bromide absorbs emissions in what wavelength?

Answer 33

300 nm

Question 34

As little as ___ ng of DNA can be detected with ethidium bromide staining?

Answer 34

5 ng

Question 35

What type of tracking dye was used in non-denaturing agarose electrophoresis during the lab?

Answer 35

Bromophenol blue

Question 36

What type of restriction enzyme results in a heterogenous collection of ends? Why?

Answer 36

Type I RE Cleaves DNA at a random position usually some distance away from their recognition sequence

Question 37

How can a linear line for the molecular weight (or length) on unknown DNA samples be made?

Answer 37

By creating a log(base pairs) by mobility (e in mm) standard curve with a known DNA marker The important part is that molecular size must be logarithmic

Question 38

How does ethanol precipitate DNA? Why is a lower concentration used for phenol removal ?

Answer 38

Ethanol is a not as polar as water and it acts to disrupt bonding between phosphate groups and water, therefore precipitating DNA out of solution. 95% ethanol is used to precipitate nucleic acids. When they are centrifuged and then vortexed, 80% ethanol is used to wash extra salts and phenol from the broken pellet.

Question 39

What are the two components of TE buffer?

Answer 39

- Tris-HCl | - EDTA

Question 40

Why is raw wheat germ used for DNA extraction?

Answer 40

Because the nucleic acids are not denatured or destroyed by cooking.

Question 41

What are the components of type IV gel loading buffer?

Answer 41

- Bromophenol blue | - Sucrose in water

Question 42

There are five types of gel loading buffer, which one should be stored at 4 degrees? What should all the rest be stored at?

Answer 42

Type V is stored at 4 degrees The rest are stored in room temperature

Question 43

Types I-V gel loading buffers are for?

Answer 43

Non-denaturing agarose gels only

Question 44

What are the components of TBE?

Answer 44

- Tris-HCl - Boric acid - EDTA

Question 45

Is agarose polymerous or crystalline?

Answer 45

Crystalline

Question 46

What are the components of restriction buffer?

Answer 46

- NaCl - Tris-HCl - MgCl2

Question 47

What are the components of a stop solution (for termination of restriction endonuclease activity)?

Answer 47

- 6X gel loading buffer type IV (bromophenol blue, sucrose) | - EDTA

Question 48

What are the components of react 2, 10X restriction buffer used in the in vitro transcription experiment?

Answer 48

- Tris-HCl - NaCl - MgCl2

Question 49

What are the components of AgCu/buffer?

Answer 49

- Ribonucleotides In buffer: - Tris-HCl - MgCl2 - Spermidine-HCl - NaCl - DTT (Dithiothreitol - redox reagent)

Question 50

How can saline citrate solution by used instead of TE buffer to prepare nucleic acid solutions when it doesn't have any EDTA?

Answer 50

Citrate forms a sodium shell around phosphate groups in DNA and inactivates nucleases that would break down the nucleic acids

Question 51

What is EDTA an abbreviation for and what does it do?

Answer 51

Ethylenediaminetetraacetic acid It is a chelating agent that can sequester magnesium ions and stop nuclease activity

Question 52

Tris-borate-EDTA is what?

Answer 52

TBE electrophoresis running buffer

Question 53

Why is it important to wait until the molten agarose gel has cooled to almost room temperature before pouring?

Answer 53

This will result in a gel with a more uniform pore size and prevent warping of the gel apparatus.

Question 54

Why is HindIII digested lambda DNA heated before being loaded onto a gel?

Answer 54

In its native state, complementary overhangs anneal to each other. This clump of DNA is inseparable on a gel unless the annealed regions are melted.

Question 55

How is phenol dangerous?

Answer 55

It is toxic by skin absorption. It can cause burns

Question 56

Where is the precipitation threshold of nucleic acids in ethanol?

Answer 56

Around 60% ethanol

Question 57

How can you determine if all the phenol has been removed from a nucleic acid pellet by rinsing with 80% ethanol?

Answer 57

By sniffing the pellet for phenol

Question 58

What ia a nucleic acid pellet dissolved in?

Answer 58

TE buffer

Question 59

What are the components of high MW RNA?

Answer 59

- 18S rRNA - 28S rRNA (26S + 5.8S) - mRNA (many sizes)

Question 60

DNA samples are stable under what temperature?

Answer 60

4 degrees celsius

Question 61

What are the components of low MW RNA?

Answer 61

- 5S rRNA | - tRNA