PCR & Agarose Gel Electrophoresis Flashcards
What’s PCR used for?
To amplify a specific sequence or sequences of DNA
What are the main ingredients of PCR?
Thermally stable DNA polymerase, pH buffer, Mg2+ (cofactor for DNA polymerase), template DNA, primers, dNTPs
What are the six basic steps of PCR?
(1) Denaturation at 95 degrees C
(2) Continued denaturation at 95 degrees C
(3) Annealing of primers at variable temperatures
(4) Extension of DNA at 68 to 72 degrees C
(5) Final incubation (finish remaining fragments)
(6) Soak at 4 degrees C
How are PCR products visualized?
Gel electrophoresis
What does the gel in gel electrophoresis contain to stain the DNA as it migrates?
Ethidium bromide
An electric current is applied to the gel, moving DNA towards the _____ (positive or negative) electrode
Positive (DNA is negatively charged)
How are molecules sorted in gel electrophoresis?
Size
How are the bands of a gel visualized?
Under UV light
What material makes up the gel?
Agarose
What is agarose?
A linear polysaccharide polymer extracted from seaweed
Agarose is a linear polysaccharide polymer extracted from a species of seaweed called _________________
Agarobiose
What’s unique about the structure of agarose?
It contains a repeated series of pentose rings that form porous matrices as it solidifes
What are DNA loading buffers?
DNA loading buffers are used for loading DNA samples onto agarose for gel electrophoresis. DNA loading buffers contains a coloured dye and a density agent. The density agent serves to enhance the density of the DNA sample allowing the DNA to sink into the bottom of the well. The dye adds visibility to the DNA sample and also serves as a tracking dye allowing the user to monitor the DNA migration during electrophoresis.
What are agarose gels made in?
TAE
What does TAE stand for?
Tris-acetate-EDTA
What is tris-acetate-EDTA?
A buffer used to make agarose gels
What is TAE?
TAE buffer is a buffer solution containing a mixture of Tris base, acetic acid and EDTA.
___________ is a buffer solution containing a mixture of Tris base, acetic acid and EDTA.
TAE or tris-acetate-EDTA
What two components make up TAE?
Tris-acetate buffer
EDTA
What’s the role of tris-acetate in TAE?
Buffer
What’s the role of EDTA in TAE?
Sequesters divalent cations
EDTA is a ________________ agent, binding to divalent cations like _____
Chelating
Mg2+
How many grams of agarose are needed for a 50 mL solution of 2% agarose?
A 1% solution = 1 g agarose/100 mL solution
A 2% solution = 2 g agarose/100 mL solution
Therefore 2 g /100 mL = x / 50 mL
x = 1 g agarose
What does it mean when we say that a stock solution is 50X?
“X” refers to a concentration version, usually of a buffer, which is cheaper to make and easier to store; it means that to get to a 1X dilution, the solution must be diluted 50-fold
You have a 50X stock solution but need 1 L or 1X stock solution. How much stock do you use? How much water?
C1V1 = C2V2
50X x X = 1X x 1 L
x = 0.02 L of stock; 0.08 L of water
What does TBE stand for?
Tris-borate-EDTA
Why is TBE not used for experiments requiring enzymatics?
Because borate is an inhibitor of many enzymes
What is TRIS?
Tris is tris-(hydroxymethyl)aminomethane
What’s the purpose of TRIS?
To protect DNA from hydrolysis
Is tris(hydroxymethyl)aminomethane a strong base or acid?
Base
Are TAE and TBE strong acids or bases?
Acids
When is it more appropriate to use a TAE buffer?
When completing enzymatic experiments
When is it more appropriate to use a TBE buffer?
When completing longer runs and requiring sharper banding patters (separation of smaller pieces of DNA)
How do we visualize DNA?
Ethidium bromide (EtBr)
What are intercalating agents?
Intercalating agents are hydrophobic heterocyclic ring molecules that resemble the ring structure of base pairs, and include ethidium bromide, acridine orange, and actinomycin D. Insertion of these agents distorts the DNA double helix, thereby interfering with DNA replication, transcription, and repair.
Why must gloves be worn when handling ethidium bromide or other gel dyes?
EtBr is a potent mutagen (can cause genetic damage), and moderately toxic after an acute exposure. EtBr can be absorbed through skin, so it is important to avoid any direct contact with the chemical. The powder form is considered an irritant to the upper respiratory tract, eyes, and skin.
What are two other common dyes for visualization other than ethidium bromide?
Fluorescent dyes (e.g., SYBR green) Actinomycin D
How does ethidium bromide function as an intercalating agent?
It becomes stacked in between DNA base pairs
How does actinomycin D enable visualization?
Its distortion of DNA results in changed UV absorbance levels that can be analyzed via microscopy and flow cytometry
What are the four basic steps to gel electrophoresis?
(1) DNA sample loading in gel loading buffer
(2) Application of electrical field
(3) DNA separates
(4) UV transillumination and documentation
What must occur prior to the four basic steps of gel electrophoresis?
The agarose mixture must be boiled, poured, and solidified; once solidified, it must be submerged in a buffer
Why do we need dyes?
DNA dyes track gel progress and do not directly interact with DNA
______________ and their migration allow us to approximate where DNA may be on the gel
Dyes
What type of dye did we use?
Orange G
Orange G loading dye migrates like a _________________ in gels ranging from 0.8% to 2.5% agarose because of the dye’s molecular weight
30 to 50 bp
