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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
____ is the most selective protein isolation method
Affinity chromatography
26
How can you determine the amount of desired protein present?
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
What is specific activity?
The ratio of amount of enzyme activity that is present in a sample to the amount of protein present
28
What is specific activity used to evaluate?
Purity; higher specific activity = purer sample
29
SDS-PAGE separates proteins based on _____
The length of their polypeptide chains
30
How many bands should a pure sample produce on SDS-PAGE?
Only one
31
In ion-exchange chromatography, separation occurs based on _____
The electrostatic attraction between oppositely charged ions
32
How do you calculate total lysozyme activity?
[activity] * volume
33
How do you calculate purification factor?
Specific activity of fraction / specific activity of FBEW
34
What is used to solidify an SDS-PAGE gel?
TEMED
35
How are proteins converted from their native state into long rods coated with negative charge?
By boiling the protein in loading buffer
36
What is found in loading buffer and what does each component do?
- Beta-mercaptoethanol - reduces disulfide bonds - Sodium dodecyl sulfate - provides the coating of negative charge - Bromophenol blue - stains the sample
37
Chlorophylls a and b strongly absorb light in the ____ and ____ regions
- Violet/blue | - Orange/red
38
What is the difference between chlorophyll a and b?
- Chlorophyll a has -CH3 | - Chlorophyll b has -CHO
39
Carotenes and xanthophylls are _____
Non-green accessory pigments
40
What colour are beta-carotene and xanthophylls in solution?
Yellow
41
Name the pigments of the chloroplast from least polar to most polar
- Beta-carotene - Xanthophylls - Chlorophyll a - Chlorophyll b
42
Which will migrate more quickly: a solute molecule that spends more time in the mobile phase or in the stationary phase?
Mobile phase
43
What makes up the stationary phase and is it polar or non-polar?
- Water | - Polar
44
What makes up the mobile phase and is it polar or non-polar?
A non-polar solvent
45
Will molecules that are more or less polar travel more quickly and why?
- Less polar | - Spend more time in the mobile phase
46
What are sodium sulfate crystals used for?
To remove excess water
47
How do you calculate Rf value?
Solute migration distance / solvent migration distance
48
What divides the interior region of a chloroplast into 2 regions and what are the 2 regions?
- Thylakoid membrane | - Stroma and lumen
49
Where do light reactions occur?
Stroma
50
What is the final electron acceptor for non-cyclic electron flow?
NADP, which is reduced to NADPH
51
___ DCPIP strongly absorbs light at 600 nm, while the ____ form doesn't
- Oxidized | - Reduced
52
How do you calculate concentration of chlorophyll using only absorbance?
A652 / 1.15
53
What is the regulatory function of pyruvate kinase?
Coordination of glycolysis and gluconeogenesis
54
What are the 4 isozymes of pyruvate kinase in mammals?
- PKL - PKR - PKM1 - PKM2
55
What does PKL do and where is it found?
- mRNA splicing variant of the PKLR gene | - Found in liver
56
What does PKR do and where is it found?
- mRNA splicing variant of the PKLR gene | - Expressed by RBC's
57
What do PKM1 and PKM2 do?
Splicing variants of the PKM gene
58
Where is PKM1 found?
- Expressed by tissues with high metabolic demands | - Found in muscle, heart, and brain
59
Where is PKM2 found?
- Expressed during embryonic development and in cancer cells | - Found in intestinal epithelium, white adipose tissue, and lungs
60
Do NADH or NAD+ absorb light at 340 nm?
NADH

Biochem 2 (40 decks)

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