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2) Biochemistry II > Lab 5: DNA Gel Electrophoresis > Flashcards

Lab 5: DNA Gel Electrophoresis Flashcards

1
Q

DNA Gel Electrophoresis may be used as a preparative technique for what other methods?

A
  • Mass spectrometry
  • RFLP
  • PCR
  • Cloning
  • DNA sequencing
  • Southern blotting
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2
Q

Define electrophoresis.

A

The electromotive force that is used to move the molecules through the gel matrix

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3
Q

Gel electrophoresis separates on the basis of what?

A

Size (number of base pairs)

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4
Q

What is the charge of nucleic acids? Where will they move?

A
  • Nucleic acids have a net negative charge

- Move from negative electrodes located near the top of the gel to positive electrodes at the bottom

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5
Q

Migration of the fragments in an electrical field move at a rate than is inversely proportional to what?

A

Log10 of their size

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6
Q

What is agarose? How can it form a gel?

A
  • Long chain polysaccharide isolated from seaweed

- Heated in a buffer solution and then cooled to form a matrix (gel) with a buffer solution trapped inside

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7
Q

What is the function of the porous lattice in the buffer solution?

A
  • Allows nucleic acids to slip through the lattice holes to move toward the positive pole
  • Larger molecules are slowed down since they can be trapped in the pores
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8
Q

Other than size, what else can affect the migration of nucleic acids?

A
  • Conformation (ss or ds)

- Charge

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9
Q

What is agarose concentration normally?

A

0.75 to 2%

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10
Q

How does the pore size change as a function of high percentage gels? What are they used for?

A
  • Higher percentage gels have smaller pores, which retards nucleic acid movement
  • Allows the separation of small fragments
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11
Q

How does the pore size change as a function of high percentage low? What are they used for?

A
  • Larger pores

- Allows the separation of large fragments

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12
Q

What is the function of a comb?

A

Used to create wells in the gel to allow for the loading of nucleic acids

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13
Q

What does the well size determine?

A

How much nucleic acid can be loaded

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14
Q

What is the most common buffer used in gel electrophoresis?

A

Tris Borate EDTA (TBE)

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15
Q

What kind of condition does the Tris maintain?

A

Slightly basic (pH 7.3)

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16
Q

What is the function of EDTA?

A

Prevents enzymatic degradation of nucleic acids as it chelates magnesium ions

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17
Q

What does the loading dye contain?

A

Glycerol

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18
Q

What is the function of glycerol?

A

The high density of glycerol aids the nucleic acid solution in settling in the well

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19
Q

What is the function of the tracking dye?

A
  • The dye moves more quickly than the nucleic acids and allows the tracking of their movements
  • Used as an indicator to alert researchers to power off the electrophoresis before the invisible nucleic acid runs off
  • Also, makes the solution more visible
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20
Q

What is a ladder?

A

A solution of known fragment sizes, which is a standard used to compare sizes of unknown nucleic acids

21
Q

What is commonly used to visualize nucleic acids? Why?

A
  • Ethidium bromide

- Fluoresces when exposed to UV light and exhibits a vivid red-orange colour when bound to nucleic acids

22
Q

What is used to visualize nucleic acid bands after running the gel?

A

UV-transilluminator

23
Q

Why is bromide dangerous?

A

Carcinogen and a mutagen

24
Q

What dyes have been developed to replace Ethidium Bromide?

A

SafeView (expensive, most labs still utilize Ethidium Bromide)

25
Q

What nucleic acids bond together in DNA?

A
  • Cytosine with Guanine

- Adenine with Thymine

26
Q

What does RNA contain instead of Thymine?

A

Uracil

27
Q

What was the DNA sample extracted from?

A

From the CHD1 (chromodomain-helicase-DNA-binding protein) gene of a chicken embryo

28
Q

How do the sex chromosomes in chickens differ?

A
  • Females: Heterogametic (ZW)

- Males: Homogametic (ZZ)

29
Q

How does the Z chromosome compare to the W?

A
  • Z is larger (376 base pairs)

- W (360 base pairs)

30
Q

How do male and female chickens differ in gel electrophoresis?

A
  • Females: two bands

- Males: one band (ZZ stacked one on top of the other)

31
Q

What is added to an Eppendorf tube?

A
  • Taq polymerase
  • dNTPs
  • Upstream and downstream primers
  • DNA template
  • Topped to 50mL with nuclease free water
32
Q

What are the three steps of PCR?

A

1) Denaturation
2) Primer annealing
3) Primer extension

33
Q

What is the first step of PCR? What is the temperature? For how long is it held?

A
  • Temp is increased to 95oC for 30 seconds

- Denatures the dsDNA to ssDNA

34
Q

What is the second step of PCR? What is the temperature? For how long is it held?

A
  • Temp is cooled to 45-68oC for 15-60 seconds
  • Allows for hybridization to occur, in which primers are added to the 3’ end of a strand, and the 3’ end of the complementary strand
35
Q

What is the third step of PCR? What is the temperature? For how long is it held?

A
  • Temp is increased to 72oC for 1min/kb

- Taq polymerase attaches dNTPs to the primers of the strands (elongation)

36
Q

What happens after 25-35 cycles of PCR?

A
  • The solution is lowered to 68oC for 5 minutes for the final extension
  • The solution is then held at 4oC
37
Q

What equation illustrates the quantity of DNA at the end of PCR cycling?

A

2^n (n is the number of cycles)

38
Q

Why is PCR kept under 35 cycles?

A
  • The exponential amplification tends to decrease after due to the degradation of dNTPs
  • The reduction of the activity of the Taq polymerase
39
Q

Why is PCR kept over 25 cycles?

A

To amplify to a suitable level to be able to properly analyze small amounts of the sample, used in forensic analysis, genetic testing, infectious disease diagnosis, analysis of ancient DNA, etc.

40
Q

What is Taq polymerase extracted from?

A

Enzyme from Thermus aquaticus, a thermophilic bacteria that lives in hot springs and tolerates high temperatures

41
Q

Why is Taq polymerase used over other polymerase enzymes?

A

Taq polymerase removes the need to add a new enzyme, such as the DNA polymerase from E. coli that was once used, following the high-heat procedures.

42
Q

What was the extraction buffer composed of?

A

Soap, sea salt, water

43
Q

What was the function of soap in the extraction buffer?

A
  • Amphipathic soap

- Dissolves the membranes and separates them from DNA

44
Q

What breaks the strawberry cells?

A

When the strawberries are squished

45
Q

What breaks the cell and nuclear membranes in the strawberry cells?

A

Soap

46
Q

What dissolves the proteins bound to the nucleic acids in the strawberry cells?

A

Salt

47
Q

What is DNA soluble in? What is DNA insoluble in?

A
  • Soluble in water

- Insoluble in ethanol

48
Q

What happens when you pour ethanol into a DNA solution?

A

Allows DNA to precipitate out of the aqueous layer and rise to the ethanol layer, which is less dense

49
Q

Why was the ethanol cold?

A

As extracted DNA from the nucleus is vulnerable to DNase enzymes, cold ethanol (cold temperatures slow down reactions) was used to protect the DNA from breakdown reactions and, thus, produce a higher yield of extractible DNA.

2) Biochemistry II (23 decks)

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