Lab 5: DNA Gel Electrophoresis

Question 1

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

Answer 1

• Mass spectrometry
• RFLP
• PCR
• Cloning
• DNA sequencing
• Southern blotting

Question 2

Define electrophoresis.

Answer 2

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

Question 3

Gel electrophoresis separates on the basis of what?

Answer 3

Size (number of base pairs)

Question 4

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

Answer 4

• Nucleic acids have a net negative charge

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

Question 5

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

Answer 5

Log10 of their size

Question 6

What is agarose? How can it form a gel?

Answer 6

• 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

Question 7

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

Answer 7

• 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

Question 8

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

Answer 8

• Conformation (ss or ds)

- Charge

Question 9

What is agarose concentration normally?

Answer 9

0.75 to 2%

Question 10

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

Answer 10

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

Question 11

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

Answer 11

• Larger pores

- Allows the separation of large fragments

Question 12

What is the function of a comb?

Answer 12

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

Question 13

What does the well size determine?

Answer 13

How much nucleic acid can be loaded

Question 14

What is the most common buffer used in gel electrophoresis?

Answer 14

Tris Borate EDTA (TBE)

Question 15

What kind of condition does the Tris maintain?

Answer 15

Slightly basic (pH 7.3)

Question 16

What is the function of EDTA?

Answer 16

Prevents enzymatic degradation of nucleic acids as it chelates magnesium ions

Question 17

What does the loading dye contain?

Answer 17

Glycerol

Question 18

What is the function of glycerol?

Answer 18

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

Question 19

What is the function of the tracking dye?

Answer 19

• 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

Question 20

What is a ladder?

Answer 20

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

Question 21

What is commonly used to visualize nucleic acids? Why?

Answer 21

• Ethidium bromide

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

Question 22

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

Answer 22

UV-transilluminator

Question 23

Why is bromide dangerous?

Answer 23

Carcinogen and a mutagen

Question 24

What dyes have been developed to replace Ethidium Bromide?

Answer 24

SafeView (expensive, most labs still utilize Ethidium Bromide)

Question 25

What nucleic acids bond together in DNA?

Answer 25

- Cytosine with Guanine | - Adenine with Thymine

Question 26

What does RNA contain instead of Thymine?

Answer 26

Uracil

Question 27

What was the DNA sample extracted from?

Answer 27

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

Question 28

How do the sex chromosomes in chickens differ?

Answer 28

- Females: Heterogametic (ZW) | - Males: Homogametic (ZZ)

Question 29

How does the Z chromosome compare to the W?

Answer 29

- Z is larger (376 base pairs) | - W (360 base pairs)

Question 30

How do male and female chickens differ in gel electrophoresis?

Answer 30

- Females: two bands | - Males: one band (ZZ stacked one on top of the other)

Question 31

What is added to an Eppendorf tube?

Answer 31

- Taq polymerase - dNTPs - Upstream and downstream primers - DNA template - Topped to 50mL with nuclease free water

Question 32

What are the three steps of PCR?

Answer 32

1) Denaturation 2) Primer annealing 3) Primer extension

Question 33

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

Answer 33

- Temp is increased to 95oC for 30 seconds | - Denatures the dsDNA to ssDNA

Question 34

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

Answer 34

- 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

Question 35

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

Answer 35

- Temp is increased to 72oC for 1min/kb | - Taq polymerase attaches dNTPs to the primers of the strands (elongation)

Question 36

What happens after 25-35 cycles of PCR?

Answer 36

- The solution is lowered to 68oC for 5 minutes for the final extension - The solution is then held at 4oC

Question 37

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

Answer 37

2^n (n is the number of cycles)

Question 38

Why is PCR kept under 35 cycles?

Answer 38

- The exponential amplification tends to decrease after due to the degradation of dNTPs - The reduction of the activity of the Taq polymerase

Question 39

Why is PCR kept over 25 cycles?

Answer 39

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.

Question 40

What is Taq polymerase extracted from?

Answer 40

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

Question 41

Why is Taq polymerase used over other polymerase enzymes?

Answer 41

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.

Question 42

What was the extraction buffer composed of?

Answer 42

Soap, sea salt, water

Question 43

What was the function of soap in the extraction buffer?

Answer 43

- Amphipathic soap | - Dissolves the membranes and separates them from DNA

Question 44

What breaks the strawberry cells?

Answer 44

When the strawberries are squished

Question 45

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

Answer 45

Soap

Question 46

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

Answer 46

Salt

Question 47

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

Answer 47

- Soluble in water | - Insoluble in ethanol

Question 48

What happens when you pour ethanol into a DNA solution?

Answer 48

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

Question 49

Why was the ethanol cold?

Answer 49

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.