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molecular lab > PCR & Agarose Gel Electrophoresis > Flashcards

PCR & Agarose Gel Electrophoresis Flashcards

1
Q

What’s PCR used for?

A

To amplify a specific sequence or sequences of DNA

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2
Q

What are the main ingredients of PCR?

A

Thermally stable DNA polymerase, pH buffer, Mg2+ (cofactor for DNA polymerase), template DNA, primers, dNTPs

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3
Q

What are the six basic steps of PCR?

A

(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

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4
Q

How are PCR products visualized?

A

Gel electrophoresis

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5
Q

What does the gel in gel electrophoresis contain to stain the DNA as it migrates?

A

Ethidium bromide

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6
Q

An electric current is applied to the gel, moving DNA towards the _____ (positive or negative) electrode

A

Positive (DNA is negatively charged)

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7
Q

How are molecules sorted in gel electrophoresis?

A

Size

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8
Q

How are the bands of a gel visualized?

A

Under UV light

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9
Q

What material makes up the gel?

A

Agarose

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10
Q

What is agarose?

A

A linear polysaccharide polymer extracted from seaweed

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11
Q

Agarose is a linear polysaccharide polymer extracted from a species of seaweed called _________________

A

Agarobiose

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12
Q

What’s unique about the structure of agarose?

A

It contains a repeated series of pentose rings that form porous matrices as it solidifes

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13
Q

What are DNA loading buffers?

A

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.

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14
Q

What are agarose gels made in?

A

TAE

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15
Q

What does TAE stand for?

A

Tris-acetate-EDTA

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16
Q

What is tris-acetate-EDTA?

A

A buffer used to make agarose gels

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17
Q

What is TAE?

A

TAE buffer is a buffer solution containing a mixture of Tris base, acetic acid and EDTA.

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18
Q

___________ is a buffer solution containing a mixture of Tris base, acetic acid and EDTA.

A

TAE or tris-acetate-EDTA

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19
Q

What two components make up TAE?

A

Tris-acetate buffer

EDTA

20
Q

What’s the role of tris-acetate in TAE?

A

Buffer

21
Q

What’s the role of EDTA in TAE?

A

Sequesters divalent cations

22
Q

EDTA is a ________________ agent, binding to divalent cations like _____

A

Chelating

Mg2+

23
Q

How many grams of agarose are needed for a 50 mL solution of 2% agarose?

A

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

24
Q

What does it mean when we say that a stock solution is 50X?

A

“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

25
Q

You have a 50X stock solution but need 1 L or 1X stock solution. How much stock do you use? How much water?

A

C1V1 = C2V2
50X x X = 1X x 1 L
x = 0.02 L of stock; 0.08 L of water

26
Q

What does TBE stand for?

A

Tris-borate-EDTA

27
Q

Why is TBE not used for experiments requiring enzymatics?

A

Because borate is an inhibitor of many enzymes

28
Q

What is TRIS?

A

Tris is tris-(hydroxymethyl)aminomethane

29
Q

What’s the purpose of TRIS?

A

To protect DNA from hydrolysis

30
Q

Is tris(hydroxymethyl)aminomethane a strong base or acid?

A

Base

31
Q

Are TAE and TBE strong acids or bases?

A

Acids

32
Q

When is it more appropriate to use a TAE buffer?

A

When completing enzymatic experiments

33
Q

When is it more appropriate to use a TBE buffer?

A

When completing longer runs and requiring sharper banding patters (separation of smaller pieces of DNA)

34
Q

How do we visualize DNA?

A

Ethidium bromide (EtBr)

35
Q

What are intercalating agents?

A

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.

36
Q

Why must gloves be worn when handling ethidium bromide or other gel dyes?

A

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.

37
Q

What are two other common dyes for visualization other than ethidium bromide?

A 
Fluorescent dyes (e.g., SYBR green)
Actinomycin D
38
Q

How does ethidium bromide function as an intercalating agent?

A

It becomes stacked in between DNA base pairs

39
Q

How does actinomycin D enable visualization?

A

Its distortion of DNA results in changed UV absorbance levels that can be analyzed via microscopy and flow cytometry

40
Q

What are the four basic steps to gel electrophoresis?

A

(1) DNA sample loading in gel loading buffer
(2) Application of electrical field
(3) DNA separates
(4) UV transillumination and documentation

41
Q

What must occur prior to the four basic steps of gel electrophoresis?

A

The agarose mixture must be boiled, poured, and solidified; once solidified, it must be submerged in a buffer

42
Q

Why do we need dyes?

A

DNA dyes track gel progress and do not directly interact with DNA

43
Q

______________ and their migration allow us to approximate where DNA may be on the gel

A

Dyes

44
Q

What type of dye did we use?

A

Orange G

45
Q

Orange G loading dye migrates like a _________________ in gels ranging from 0.8% to 2.5% agarose because of the dye’s molecular weight

A

30 to 50 bp

molecular lab (4 decks)

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