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Nucleic Acid Lab > Theory and Technique > Flashcards

Theory and Technique Flashcards

1
Q

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

A

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

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2
Q

Why is RNA hard to extract from cells?

A

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

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3
Q

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

A
  • Highly purified protease solutions that degrade protein
  • Strong salts to dissolve nucleic acids and precipitate denatured protein
  • Phenol can denature protein
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4
Q

Where are ribonucleases found?

A

In most living cells and in breath and perspiration

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5
Q

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

A

To denature ribonucleases that may degrade RNA

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6
Q

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

A

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

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7
Q

Why are nucleic acid extractions carried out on ice?

A

To minimize nuclease activity

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8
Q

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

A

8-hydroxyquinoline

Chelating agent that soaks up divalent cations

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9
Q

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

A
  • Nucleic acids (upper aqueous layer)
  • Denatured protein (middle phenol-aqueous interface phase)
  • Hydrophobic cell components (lower phenol phase)
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10
Q

Phenol extractions are completed until?

A

No further protein is observed at the phenol-aqueous interface

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11
Q

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

A
  • Absolute (near 100%) ethanol precipitates nucleic acids

- 80% removes phenol from pellet

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12
Q

How is high MW RNA precipitated?

A

Strong solutions of sodium chloride

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13
Q

How is DNA precipitated?

A

10% w/v polyethylene glycol (PEG)

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14
Q

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?

A

The facilitate the aggregation of charged nucleic acid molecuels

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15
Q

In which order are nucleic acid fractions fractionated?

A
  • High MW RNA
  • DNA
  • Low MW RNA
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16
Q

What are the components of a nucleic acid extractino buffer?

A
  • Tris-HCL
  • EDTA-Na
  • Sarkosyl
  • Nacl
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17
Q

Purines and pyrimidines absorb light strongly at ____ nm

A

260 nm

True for both RNA and DNA

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18
Q

Proteins exhibit a strong absorption at ____ nm. Why?

A

280 nm

Aromatic amino acid content (tyrosine and tryptophan)

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19
Q

Polysaccharides show strong absorbances around ___ nm

A

230

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20
Q

For pure nucleic acids 260/280 = ?

A

1.8

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21
Q

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

A

Protein

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22
Q

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

A

Buffer contamination

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23
Q

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

A

A260

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24
Q

What is a specific extinction coefficient defined as?

A

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

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25
Q

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

A

The Beer-Lambert law

A = KcL

A = absorbance
K = Constant
c = concentration
L = Length of light path (cm)
26
Q

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

A

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

Unit conversions are necessary

27
Q

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

A

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

28
Q

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

A

DNA: 50
RNA: 40
Oligonucleotides: 33

29
Q

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

A

For accurate measurements of 260/280 purity check

30
Q

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

A

280 nm

31
Q

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

A
  • Keeps the gel cool as it runs

- Contains ions that will conduct the electricity

32
Q

What are three purposes for gel loading buffer?

A
  • 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
33
Q

Ethidium bromide absorbs emissions in what wavelength?

A

300 nm

34
Q

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

A

5 ng

35
Q

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

A

Bromophenol blue

36
Q

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

A

Type I RE

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

37
Q

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

A

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

38
Q

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

A

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.

39
Q

What are the two components of TE buffer?

A
  • Tris-HCl

- EDTA

40
Q

Why is raw wheat germ used for DNA extraction?

A

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

41
Q

What are the components of type IV gel loading buffer?

A
  • Bromophenol blue

- Sucrose in water

42
Q

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

A

Type V is stored at 4 degrees

The rest are stored in room temperature

43
Q

Types I-V gel loading buffers are for?

A

Non-denaturing agarose gels only

44
Q

What are the components of TBE?

A
  • Tris-HCl
  • Boric acid
  • EDTA
45
Q

Is agarose polymerous or crystalline?

A

Crystalline

46
Q

What are the components of restriction buffer?

A
  • NaCl
  • Tris-HCl
  • MgCl2
47
Q

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

A
  • 6X gel loading buffer type IV (bromophenol blue, sucrose)

- EDTA

48
Q

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

A
  • Tris-HCl
  • NaCl
  • MgCl2
49
Q

What are the components of AgCu/buffer?

A
  • Ribonucleotides

In buffer:

  • Tris-HCl
  • MgCl2
  • Spermidine-HCl
  • NaCl
  • DTT (Dithiothreitol - redox reagent)
50
Q

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

A

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

51
Q

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

A

Ethylenediaminetetraacetic acid

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

52
Q

Tris-borate-EDTA is what?

A

TBE electrophoresis running buffer

53
Q

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

A

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

54
Q

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

A

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.

55
Q

How is phenol dangerous?

A

It is toxic by skin absorption. It can cause burns

56
Q

Where is the precipitation threshold of nucleic acids in ethanol?

A

Around 60% ethanol

57
Q

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

A

By sniffing the pellet for phenol

58
Q

What ia a nucleic acid pellet dissolved in?

A

TE buffer

59
Q

What are the components of high MW RNA?

A
  • 18S rRNA
  • 28S rRNA (26S + 5.8S)
  • mRNA (many sizes)
60
Q

DNA samples are stable under what temperature?

A

4 degrees celsius

61
Q

What are the components of low MW RNA?

A
  • 5S rRNA

- tRNA

Nucleic Acid Lab (2 decks)

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