(M) Gel Electrophoresis (LAB) Flashcards by Nathalie Grace Castillo

This technique is used:
- to separate (sometimes purify) macromolecules especially proteins and nucleic acids
- to determine the presence of DNA and separate them from each other based on their size
- in DNA fingerprinting
- to analyze PCR results

Gel Electrophoresis

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T or F: Macromolecules don’t differ in size, only conformation

False (they differ in both)

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When charged molecules are within an electric field, they migrate towards an area of what charge?

Opposite

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T or F: Proteins can have only have a negative charge because of their phosphate backbone

False (that description is for nucleic acids)

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This material in gel electrophoresis:
- has ions to carry a current
- maintains the pH at a relatively constant value

Buffer

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This is a polysaccharide extracted from seaweed, is non-toxic, and is to be used at concentrations of 0.5% to 2%

Agarose

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T or F: More agarose = stiffer gel

True

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T or F: Agarose has a small range of separation but high resolving power

False (polyacrylamide; the opposite is for agarose)

Resolving power is the ability to differentiate two lines or points in an object

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What is the range of base pairs agarose can separate?

200 - 50,000

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This is a cross-linked polymer of acrylamide, is to be used with concentrations from 3.5% to 20%, and is more annoying to prepare than the other kind of gel

Polyacrylamide

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Why are polyacrylamide gels more annoying to prepare than agarose?

O2 inhibits polymerization (they need to be poured between glass plates or cylinders)

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T or F: Polyacrylamide is a potent neurotoxin

False (acrylamide is the potent neurotoxin while polyacrylamide is harmless, although some acrylamide can be left out)

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What is the range of base pairs that polyacrylamide can separate?

Less than 500 only (due to its low separation range)

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This is a GENERAL method of separating electrically charged substances in a mixture (DNA, RNA, proteins, etc.) where the sample is placed on a supporting medium to which an electrical field is applied

Electrophoresis (lang)

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Which is the positive and negative terminal between anode and cathode?

Cathode - negative
Anode - positive

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Which terminal will positively charged molecules migrate to?

Cathode (negative)

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Which terminal will negatively charged molecules migrate to?

Anode (positive)

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T or F: The speed of migration depends on charge, size, and shape

True

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The General Parts of Electrophoresis:
→ responsible for allowing the charged molecules to move

→ the voltage depends on the protocol

Voltage or power supply

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The General Parts of Electrophoresis:
→ a fluid/aqueous solution around the supporting medium and is present within the chamber

Buffer system/solution

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He is the Father of Electrophoresis who did pioneer work on moving boundary electrophoresis in 1930 and later developed a zone method for biomolecule purification

Arne Tiselius

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T or F: Arne Tiselius, despite his contributions, did not win a Nobel prize

False (he did)

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Arne Tiselius used this specimen for his research on electrophoresis

Serum

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These three made tools for DNA gel electrophoresis in the 1970s and used agarose gel extracted from seaweed

Phil Sharp, Joe Sambrooke, and Bill Sugden

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T or F: The gel material acts as a “molecular sieve” by retarding/completely obstructing the movement of micromolecules and allowing macromolecules to migrate freely

False (obstructs MACROmolecules while allowing MICROmolecules to move freely)

Which among the 2 gels separates large molecules?

Agarose (polyacrylamide is for small molecules)

Which among the gels are casted horizontally?

Agarose (polyacrylamide is casted vertically)

T or F: Polyacrylamide is used for DNA or protein separations

True (due to its high resolving power) *Agarose is commonly used for DNA separation only

When can staining be done when using polyacrylamide gel?

After pouring the gel *Agarose staining can be done before or during the pouring

At what temperature does agarose begin to solidify and become a gel?

40ºC

This is a linear carbohydrate polymer composed of repeating units of agarobiose (alternating units of galactose and 3,6-anhydrogalactose)

Agarose

T or F: The gelling/solidifying property of agarose is due to polymerization

False (that is for polyacrylamide; agarose gelling is due to both inter- and intramolecular H bonding which shifts from random coils in a solution to chains bundled into double helices)

This piece of equipment contains black and red wires and is where the electrophoresis will occur

Electrophoresis chamber

What color are the wires for the anode and cathode?

Cathode is black Anode is red (this is where molecules will migrate towards)

T or F: The buffer already comes in diluted form prior to the procedure

False (comes in concentrated forms; you will need to dilute)

This adheres to DNA. gives off fluorescent light, and allows visual monitoring of how far the process has proceeded

Staining agent or tracking dye

This chemical is added to the gel with a final concentration of 0.5 µg/ml to facilitate visualization after electrophoresis (this intercalates between DNA and RNA bases)

Ethidium Bromide

The agarose solution is allowed to cool until what temperature before being poured into the casting tray?

60ºC

These are available in many sizes, are composed of UV-transparent plastic, and is where the gel is poured

Gel casting trays

T or F: The open ends of the casting trays can be enclosed with tape that can stay until the end of electrophoresis

False (needs to be removed before electrophoresis)

These form wells in the gel

Sample combs

What are the 2 common buffers in electrophoresis?

- Tris-acetate-EDTA (TAE) - Tris-borate-EDTA (TBE)

T or F: A loading buffer must contain something light

False (must be dense in order to sink the sample, like glycerol)

T or F: Ethidium bromide is a mutagen

True

This UV lightbox is used to visualize the ethidium bromide-stained DNA

Transilluminator

There is evidence of a current if ______ are observed coming off of the electrodes

Bubbles

Bromophenol blue migrates at the same rate as DNA fragments with how many base pairs?

300

Xylene cyanol migrates at the same rate as DNA fragments with how many base pairs?

4000

T or F: Ethidium bromide can only be incorporated after electrophoresis has took place

False (it can be incorporated during electrophoresis or afterwards by soaking it in dilute ethidium bromide solution)

T or F: DNA will diffuse within the gel overtime so examination/photography should take place immediately after the process is done

True

- This is aka a molecular weight marker - Is placed on the first or middle well - Serves as a basis for the estimation of DNA size or the number of base pairs - Each line corresponds to DNA sizes

DNA ladder

- This is a mixture of glycerol and tracking dye to be mixed in with the sample - Glycerol adds density to the sample in order for it to sink into the well

Loading dye

Viewing the Samples: → use of a UV transilluminator to view the fluorescent DNA in the gel

DNA detection

Viewing the Samples: → dye and size must be the same to the DNA being measured

DNA quantification

Viewing the Samples: → bands appear as compact, no double or faint bonds

Evaluation of the quality of DNA

This is expressed in molecular weight equivalent to the number of base pairs

Molecular size

T or F: Small fragments travel faster and farther while large fragments travel slower due to greater frictional drag

True

T or F: Small fragments find other small holes in the gel to pass through

False (large)

Factors that Determine Migration Rate: A linear DNA fragment of a given size migrates at different rates with different concentrations of the gel

Agarose concentration

T or F: Higher concentrations of agarose facilitates separation of larger DNA fragments

False (smaller) *Lower concentrations = larger fragments *Higher concentrations = smaller fragments

Factors that Determine Migration Rate: Supercoiled circular, relaxed circular, and linear DNA of the same molecular weights will migrate at different rates

DNA conformation

What is the order in of DNA conformation that travels the fastest to slowest?

Supercoiled > Linear DS > Circular Explanation: - supercoiled DNA become smaller in size therefore they will have less frictional resistance and are more compact - when supercoiled DNA is broken due to nicks or cuts, it goes back to its normal relaxed circular conformation (this is now larger and therefore has more frictional drag) - if the circular DNA is cut, it becomes linear (this migrates at a normal speed and in accordance to size)

Factors that Determine Migration Rate: The rate of migration is directly proportional to this

Voltage

T or F: Higher voltage = faster migration while lower voltage = slower migration

True

T or F: If the voltage is too low, the gel may melt and result to sample smearing, DNA denaturation, and sample damage

False (high)

T or F: Sharper resolution of larger DNA fragments for low voltages is a con

False (pro) *The con is that slower migration may cause diffusion of DNA fragments

Factors that Determine Migration Rate: The composition and ionic strength of this affects DNA mobility

Electrophoresis buffer

If _____ is used, electrical conductivity is minimal therefore DNA migrates slower

Water

T or F: If a buffer with high ionic strength is used (10x) then electrical conductance is efficient

True (however this concentrated buffer generates more heat leading to gel melting and DNA denaturation)

Factors that Determine Migration Rate: → dyes used to stain are usually intercalating agents → addition to the gel may retard the migration rate of DNA

Presence of DNA stains in the buffer

Factors that Determine Migration Rate: Has something to do with their melting temperature

Type of agarose

This type of agarose: → gels at 35-38ºC and melts at 90-95ºC → detects DNA fragments ranging from 1,000 bps to 25,000 bps

High melting temperature (standard)

This type of agarose: → gels at 35ºC and melts at 65ºC → is used for rapid recovery of DNA from the gel

Low melting temperature