Lab 5: DNA Gel Electrophoresis Flashcards
DNA Gel Electrophoresis may be used as a preparative technique for what other methods?
- Mass spectrometry
- RFLP
- PCR
- Cloning
- DNA sequencing
- Southern blotting
Define electrophoresis.
The electromotive force that is used to move the molecules through the gel matrix
Gel electrophoresis separates on the basis of what?
Size (number of base pairs)
What is the charge of nucleic acids? Where will they move?
- Nucleic acids have a net negative charge
- Move from negative electrodes located near the top of the gel to positive electrodes at the bottom
Migration of the fragments in an electrical field move at a rate than is inversely proportional to what?
Log10 of their size
What is agarose? How can it form a gel?
- 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
What is the function of the porous lattice in the buffer solution?
- 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
Other than size, what else can affect the migration of nucleic acids?
- Conformation (ss or ds)
- Charge
What is agarose concentration normally?
0.75 to 2%
How does the pore size change as a function of high percentage gels? What are they used for?
- Higher percentage gels have smaller pores, which retards nucleic acid movement
- Allows the separation of small fragments
How does the pore size change as a function of high percentage low? What are they used for?
- Larger pores
- Allows the separation of large fragments
What is the function of a comb?
Used to create wells in the gel to allow for the loading of nucleic acids
What does the well size determine?
How much nucleic acid can be loaded
What is the most common buffer used in gel electrophoresis?
Tris Borate EDTA (TBE)
What kind of condition does the Tris maintain?
Slightly basic (pH 7.3)
What is the function of EDTA?
Prevents enzymatic degradation of nucleic acids as it chelates magnesium ions
What does the loading dye contain?
Glycerol
What is the function of glycerol?
The high density of glycerol aids the nucleic acid solution in settling in the well
What is the function of the tracking dye?
- 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
What is a ladder?
A solution of known fragment sizes, which is a standard used to compare sizes of unknown nucleic acids
What is commonly used to visualize nucleic acids? Why?
- Ethidium bromide
- Fluoresces when exposed to UV light and exhibits a vivid red-orange colour when bound to nucleic acids
What is used to visualize nucleic acid bands after running the gel?
UV-transilluminator
Why is bromide dangerous?
Carcinogen and a mutagen
What dyes have been developed to replace Ethidium Bromide?
SafeView (expensive, most labs still utilize Ethidium Bromide)
What nucleic acids bond together in DNA?
- Cytosine with Guanine
- Adenine with Thymine
What does RNA contain instead of Thymine?
Uracil
What was the DNA sample extracted from?
From the CHD1 (chromodomain-helicase-DNA-binding protein) gene of a chicken embryo
How do the sex chromosomes in chickens differ?
- Females: Heterogametic (ZW)
- Males: Homogametic (ZZ)
How does the Z chromosome compare to the W?
- Z is larger (376 base pairs)
- W (360 base pairs)
How do male and female chickens differ in gel electrophoresis?
- Females: two bands
- Males: one band (ZZ stacked one on top of the other)
What is added to an Eppendorf tube?
- Taq polymerase
- dNTPs
- Upstream and downstream primers
- DNA template
- Topped to 50mL with nuclease free water
What are the three steps of PCR?
1) Denaturation
2) Primer annealing
3) Primer extension
What is the first step of PCR? What is the temperature? For how long is it held?
- Temp is increased to 95oC for 30 seconds
- Denatures the dsDNA to ssDNA
What is the second step of PCR? What is the temperature? For how long is it held?
- 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
What is the third step of PCR? What is the temperature? For how long is it held?
- Temp is increased to 72oC for 1min/kb
- Taq polymerase attaches dNTPs to the primers of the strands (elongation)
What happens after 25-35 cycles of PCR?
- The solution is lowered to 68oC for 5 minutes for the final extension
- The solution is then held at 4oC
What equation illustrates the quantity of DNA at the end of PCR cycling?
2^n (n is the number of cycles)
Why is PCR kept under 35 cycles?
- The exponential amplification tends to decrease after due to the degradation of dNTPs
- The reduction of the activity of the Taq polymerase
Why is PCR kept over 25 cycles?
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.
What is Taq polymerase extracted from?
Enzyme from Thermus aquaticus, a thermophilic bacteria that lives in hot springs and tolerates high temperatures
Why is Taq polymerase used over other polymerase enzymes?
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.
What was the extraction buffer composed of?
Soap, sea salt, water
What was the function of soap in the extraction buffer?
- Amphipathic soap
- Dissolves the membranes and separates them from DNA
What breaks the strawberry cells?
When the strawberries are squished
What breaks the cell and nuclear membranes in the strawberry cells?
Soap
What dissolves the proteins bound to the nucleic acids in the strawberry cells?
Salt
What is DNA soluble in? What is DNA insoluble in?
- Soluble in water
- Insoluble in ethanol
What happens when you pour ethanol into a DNA solution?
Allows DNA to precipitate out of the aqueous layer and rise to the ethanol layer, which is less dense
Why was the ethanol cold?
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.
