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Biochem 2 > Lab Exam > Flashcards

Lab Exam Flashcards

1
Q

Columns for DNA purification are usually ______

A

Silica-based

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2
Q

How can DNA be eluted out of a column?

A

Altering the salt and pH conditions in the column

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3
Q

How is purification done after the cells of a tissue have been lysed?

A

Selectively degrading and precipitating non-DNA biological molecules

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4
Q

How are non-DNA biological molecules removed?

A
  • Centrifugation

- Washing them off the column

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5
Q

What temperature must DNA be stored at?

A

-20 C

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6
Q

What does isopropanol do to DNA?

A

Precipitates it

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7
Q

What does lysis buffer do?

A

Breaks down the plant cell walls

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8
Q

What does placing DNA in a 65 C dry bath do?

A

Solubilizes membranes

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9
Q

What does precipitation buffer do?

A

Precipitates proteins

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10
Q

What is the purpose of centrifugation?

A

To separate DNA from polysaccharides

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11
Q

What does elution buffer do?

A

Changes the ion interaction of the column and DNA

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12
Q

How can you determine purity of a sample of DNA?

A

Absorbance at 260 nm / absorbance at 280 nm

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13
Q

When will a sample of 50 ug/mL DNA have an absorbance of 1.0?

A

At 260 nm

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14
Q

What are restriction enzymes?

A

Proteins that cut the sugar phosphate backbone of DNA only when a specific sequence of nucleotides is present

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15
Q

What determines the size of fragments that are produced?

A
  • Sequence of DNA

- Restriction enzyme used

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16
Q

What occurs when DNA is mixed with a restriction enzyme?

A

Digestion, which is a incubation and cutting process

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17
Q

_____ is one of the simplest and most widespread methods to analyze DNA

A

Agarose gel electrophoresis

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18
Q

Define electrophoresis

A

The examination of the movement of charged particles through solution under the influence of an electric field

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19
Q

What is the electrophoresis equation?

A

v = Eq / f

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20
Q

What does the fractional coefficient represent?

A

The size and shape of the molecule

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21
Q

The amount of negative charge on a DNA molecule is proportional to ____

A

Its length

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22
Q

Average pore size _____ with increasing concentrations of agarose

A

Decreases

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23
Q

Do shorter DNA molecules travel further or shorter and why?

A

Further because they have a greater number of pores that they can migrate through

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24
Q

What is used to dye DNA for electrophoresis?

A

HydraGreen

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25
Q

____ is the most selective protein isolation method

A

Affinity chromatography

26
Q

How can you determine the amount of desired protein present?

A

Add a small fixed amount of the purification product to an excess of enzyme substrate and measure the amount of product that is formed

27
Q

What is specific activity?

A

The ratio of amount of enzyme activity that is present in a sample to the amount of protein present

28
Q

What is specific activity used to evaluate?

A

Purity; higher specific activity = purer sample

29
Q

SDS-PAGE separates proteins based on _____

A

The length of their polypeptide chains

30
Q

How many bands should a pure sample produce on SDS-PAGE?

A

Only one

31
Q

In ion-exchange chromatography, separation occurs based on _____

A

The electrostatic attraction between oppositely charged ions

32
Q

How do you calculate total lysozyme activity?

A

[activity] * volume

33
Q

How do you calculate purification factor?

A

Specific activity of fraction / specific activity of FBEW

34
Q

What is used to solidify an SDS-PAGE gel?

A

TEMED

35
Q

How are proteins converted from their native state into long rods coated with negative charge?

A

By boiling the protein in loading buffer

36
Q

What is found in loading buffer and what does each component do?

A
  • Beta-mercaptoethanol - reduces disulfide bonds
  • Sodium dodecyl sulfate - provides the coating of negative charge
  • Bromophenol blue - stains the sample
37
Q

Chlorophylls a and b strongly absorb light in the ____ and ____ regions

A
  • Violet/blue

- Orange/red

38
Q

What is the difference between chlorophyll a and b?

A
  • Chlorophyll a has -CH3

- Chlorophyll b has -CHO

39
Q

Carotenes and xanthophylls are _____

A

Non-green accessory pigments

40
Q

What colour are beta-carotene and xanthophylls in solution?

A

Yellow

41
Q

Name the pigments of the chloroplast from least polar to most polar

A
  • Beta-carotene
  • Xanthophylls
  • Chlorophyll a
  • Chlorophyll b
42
Q

Which will migrate more quickly: a solute molecule that spends more time in the mobile phase or in the stationary phase?

A

Mobile phase

43
Q

What makes up the stationary phase and is it polar or non-polar?

A
  • Water

- Polar

44
Q

What makes up the mobile phase and is it polar or non-polar?

A

A non-polar solvent

45
Q

Will molecules that are more or less polar travel more quickly and why?

A
  • Less polar

- Spend more time in the mobile phase

46
Q

What are sodium sulfate crystals used for?

A

To remove excess water

47
Q

How do you calculate Rf value?

A

Solute migration distance / solvent migration distance

48
Q

What divides the interior region of a chloroplast into 2 regions and what are the 2 regions?

A
  • Thylakoid membrane

- Stroma and lumen

49
Q

Where do light reactions occur?

A

Stroma

50
Q

What is the final electron acceptor for non-cyclic electron flow?

A

NADP, which is reduced to NADPH

51
Q

___ DCPIP strongly absorbs light at 600 nm, while the ____ form doesn’t

A
  • Oxidized

- Reduced

52
Q

How do you calculate concentration of chlorophyll using only absorbance?

A

A652 / 1.15

53
Q

What is the regulatory function of pyruvate kinase?

A

Coordination of glycolysis and gluconeogenesis

54
Q

What are the 4 isozymes of pyruvate kinase in mammals?

A
  • PKL
  • PKR
  • PKM1
  • PKM2
55
Q

What does PKL do and where is it found?

A
  • mRNA splicing variant of the PKLR gene

- Found in liver

56
Q

What does PKR do and where is it found?

A
  • mRNA splicing variant of the PKLR gene

- Expressed by RBC’s

57
Q

What do PKM1 and PKM2 do?

A

Splicing variants of the PKM gene

58
Q

Where is PKM1 found?

A
  • Expressed by tissues with high metabolic demands

- Found in muscle, heart, and brain

59
Q

Where is PKM2 found?

A
  • Expressed during embryonic development and in cancer cells

- Found in intestinal epithelium, white adipose tissue, and lungs

60
Q

Do NADH or NAD+ absorb light at 340 nm?

A

NADH

Biochem 2 (40 decks)

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