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Lab final Flashcards

1
Q

list 8 specimen types from which DNA can be isolated

A

whole blood, buffy coat, bone marrow, solid tissue, lavage fluids, bacteria/viruses, fungi, organelles (mitochondria)

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2
Q

what qualifies a specimen to be capable of DNA isolation?

A

any specimen that contains cells with nuclei

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3
Q

what are the parts of the anticoagulated blood specimen? where is the DNA found?

A

plasma, buffy coat, erythrocytes; DNA found in buffy coat

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4
Q

What is the purpose of the Chelex reagent in crude DNA extractions? Why is this important?

A

Chelex removes multivalent cations that can damage DNA.

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5
Q

Explain the purpose of each reagent in the strawberry DNA isolation procedure:

A

Shampoo: lysis the cells when the fruit is smashed in the buffer
Salt: promotes the precipitation of the DNA (removes DNA-bound proteins)
cold alcohol: DNA precipitates into the alcohol layer, DNA is insoluble in alcohol

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6
Q

What do the crude DNA extractions have in common? In what way do they vary?

A

Allow for release of DNA by cell lysis, the DNA is not purified

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7
Q

what is the difference bw extraction vs isolation?

A

isolation = DNA is cleaned up, isolated DNA is best

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8
Q

what do the letters stand for in PCI? what kind of a DNA isolation process is this?

A

phenol, chloroform, isoamyl alcohol; organic

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9
Q

what’s the first step in any DNA procedure? and what’s used?

A

lysis; detergents and proteases

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10
Q

what needs to be done if the pH needs to be lowered in an organic procedure? using what?

A

acidification; acetic acid and salt

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11
Q

in an organic isolation what is added to encourage precipitation (salts have already been added)

A

sodium acetate or ammonium acetate

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12
Q

what percentage of ethanol is added during ethanol precipitation? why is this done?

A

100%; DNA is insoluble in alcohol

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13
Q

what is the purpose of the 70% ethanol wash? what happens after this step?

A

dissolve salt with dilute alcohol; tube is inverted to air dry

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14
Q

what is the very last step of organic DNA isolation?

A

DNA is resuspended in buffer or water to dissolve DNA

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15
Q

List two reasons why the solid phase Qiagen spin column isolation method is better than the PCI method.

A

Highly purified DNA and less toxic chemicals

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16
Q

List two reasons why the PCI method is better than the Qiagen spin column isolation method.

A

molecular weight of DNA is higher; the amount and quality of DNA is higher

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17
Q

what are the 5 steps of the Qiagen method in order?

A
  1. Lysis using AL buffer and proteinase K
  2. incubation at 56 degrees
  3. addition of 100% ethanol
  4. addition of AW1/AW2 buffers
  5. Elution with AE buffer
18
Q

what is the formula for calculating DNA concentration using spectrophotometry? How is the formula different for RNA calculations?

A

(A260)(50ug/mL)(dilution factor)
(A260)(40ug/mL)(dilution factor)

19
Q

Explain why traditional spectrophotometry (not the Nanodrop) requires a DNA dilution step?

A

It is too concentrated to measure directly or in not in a volume that was measurable in a cuvette

20
Q

at what wavelength does DNA, RNA, and protein absorb light?

A

260, 260, 280 nm

21
Q

If the value for A280 is high, does that cause the purity ratio to be higher or lower?

A

lower

22
Q

what is the ideal purity ratio for DNA? what is the range?

A

1.8; 1.6-2.1

23
Q

what is an acceptable A260/A280 ratio for RNA?

A

1.7-2.3

24
Q

An isolated DNA sample gives a A260/280 ratio of 1.4. What contaminant is likely present?

A

protein; ratio is low so that means a high 280 value

25
Q

You isolate DNA and after spectrophotometry, you determine that the DNA has a concentration of 600 µg/mL and an A260/280 ratio of 1.3. What does this indicate about the quality of the DNA? Is the DNA conc accurate?

A

protein contamination; no, concentration is lower

26
Q

Why is no dilution necessary for the Nanodrop?

A

When the arm is lowered and contacts the sample, it creates a liquid column with path length defined by gap bw the two optical surfaces

27
Q

List three differences between Nanodrop spectrophotometry and fluorometry.

A
  • Fluorometry is more sensitive than spectrophotometry
  • Fluorometry is great for very small amounts of DNA
  • Fluorometry involves the binding of a fluorescent dye to the DNA
28
Q

what determines the intensity of the bands in a gel?

A

mass of DNA

29
Q

what does it mean when a gel is smeared?

A

DNA is degraded

30
Q

what indicates high quality DNA on a gel?

A

Intense, high molecular weight band with little to no smearing

31
Q

In gel electrophoresis, why does DNA move toward the positive electrode? DNA is thus resolved based on what factor?

A

DNA is negatively charged; resolved size, molecular weight, # of base pairs

32
Q

what are 4 characteristics of agarose vs polyacrylamide gel?

A

lower resolving power, very porous, better for separating large fragments, most commonly used, highest conc ~ 5

33
Q

what are 4 characteristics of polyacrylamide vs agarose gel?

A

finer resolution, protein electrophoresis, toxic components, can go higher on concentration ~20

34
Q

Which percentage agarose gel would be best to resolve two DNA fragments that are around 3000 bp in size?

A

1.5%, lower % for larger size

35
Q

Which percentage polyacrylamide gel would be best to resolve two DNA fragments that around 20 bp in size?

A

20%; higher % for small fragments

36
Q

Outline the steps for making an agarose gel, starting from agarose powder and 10X TBE.

A
  • Determine the % of agarose concentration needed
  • Dilute with buffer to proper concentration
  • Heat solution to dissolve
  • Cool to 55-60 degrees C
    add gel red to see distance traveled
  • Pour gel into casting tray
  • Allow to polymerize
  • Remove comb, prepare to load
37
Q

List the reagents/components used to make a polyacrylamide gel. Briefly state the purpose of each.

A
  • Acrylamide powder – used to make stock solution
  • Bis-acrylamide – used as a crosslinker
  • Ammonium persulfate – initiates polymerization
  • TEMED – stabilizes polymerization
38
Q

what color is the negative electrode? where would this be connected?

A

black; side with the wells

39
Q

List three purposes of loading dye for electrophoresis

A

Gives color to DNA for easier visualization
* Makes DNA denser so it sink to the bottom
* Has tracking dyes that separate during electrophoresis to indicate progress.

40
Q

What is required to visualize DNA in a gel?

A

Adding gel red + UV light

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