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Biomedical Lab > Lab 9 > Flashcards

Lab 9 Flashcards

1
Q

What is gel electrophoresis?

A

A procedure used to separate biomolecules such as DNA, RNA, or proteins using a gel matrix made of agarose or polyacrylamide

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2
Q

Separation in gel electrophoresis depends on….

A

The properties of the gel as well as the molecules to be separated

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3
Q

Gel matrix contains…

A

Pores of a uniform size through which molecules can pass through

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4
Q

Which makes larger pores - agarose or polyacrylamide?

A

Agarose

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5
Q

What determines the size of the pores in gel electrophoresis?

A

Gel concentration

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6
Q

The higher the gel concentration, the ____ the pores will be

A

Smaller

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7
Q

What molecules are separated using agarose gel? Why?

A

Nucleic acids (DNA and RNA) because they are very large molecules and agarose creates larger pores than polyacrylamide

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8
Q

Polyacrylamide is used to separate____. Why?

A

Proteins because they are generally much smaller than DNA and RNA

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9
Q

What is the most important factor for migration through the gel matrix?

A

The size of the molecules

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10
Q

Which migrate faster-large or small molecules?

A

Small

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11
Q

What is the native structure and most stable form of DNA?

A

Supercoiled

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12
Q

When coiled, DNA is very ___ and ____

A

Small and compact

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13
Q

Which moves faster through the gel - coiled DNA or DNA that has been cut by a restriction enzyme (and is now linear)

A

Coiled will move faster because its very small and compact

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14
Q

Why does DNA that has been cut with a restriction enzyme move slowly through the gel?

A

Nicked DNA fragments will be generated when DNA is cut with a restriction enzyme.
These nicked fragments will act as a hook and hold DNA to the gel

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15
Q

How is the agarose gel prepared?

A

By heating the agarose powder in the buffer

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16
Q

When preparing agarose for electrophoresis, it is best to ____ the agarose into the buffer at _____(what temp), swirl, and let it sit at least ______minutes before microwaving

A

SPRINKLE, ROOM TEMP, 1 MIN

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17
Q

Why is it best to follow this procedure?

A

This allows the agarose to hydrate first, which minimizes foaming during heating

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18
Q

The hot liquid is poured into a ——-

A

Casting tray containing a comb

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19
Q

The comb is removed from the casting tray when…

A

The gel solidifies to leave wells in the gels

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20
Q

The DNA sample is mixed with a ___ and loaded into ____

A

Mixed with a DNA LOADING BUFFER and loaded into WELLS

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21
Q

What does the DNA loading buffer contain?

A

It is a concentrate containing a tracking dye and glycerol

22
Q

Explain the colored (blue) tracking dye and how it works

A

It has a low molecular weight and runs ahead of the DNA fragments and tracks the progress of the colorless DNA through the gel

23
Q

What is the purpose of the glycerol in the DNA loading buffer?

A

It increases the density of the sample and facilitates it to fall easily in the wells and prevents it from flowing away

24
Q

What is used to determine the size of the DNA in the samples?

A

A DNA marker containing a mixture of DNA fragments of known sizes

25
Q

____ and ____ DNA are used to compare and analyze the gel observations

A

Digested (cut) and non-digested(uncut) DNA controls

26
Q

What will result in sharper bands?

A

Loading DNA in the smallest volume possible

27
Q

What do molecules need to move through the gel pores?

A

Electric current

28
Q

The higher the voltage, the ____ DNA moves

A

Faster

29
Q

DNA moves faster in high voltage, but what is the drawback to this?

A

It heats the gel and ultimately causes it to melt, denature the DNA, and decrease the resolution (separation) of the DNA fragments

30
Q

____ affects the migration of molecules through an electric field

A

Charge

31
Q

When a current is applied, positive ions (cations) will move toward the…

A

Cathode (negative electrode)

32
Q

When a current is applied, negative ions (anions) will move towards—-

A

The anode (positive electrode)

33
Q

Will molecules of the same size but different charges be the same in migration?

A

NO

34
Q

To separate molecules by size only, all the molecules must have….

A

A similar charge

35
Q

Why is water not used for preparing gels or used as a running solution?

A

Because it is not a good conductor of electricity

36
Q

Why is a running buffer containing electrolytes used?

A

To ensure the conductance of electricity through the gel

37
Q

What is the charge of DNA and RNA at a pH of 8? Why?

A

Negative charge because they both contain ionizable phosphate groups

38
Q

Why is the TAE buffer preferred to other buffers such as TBE?

A

Because it provides better resolution for fragments >4kb

39
Q

The gel is covered ____-____mm with the buffer

A

2-3mm

40
Q

Too much buffer results in….

A

Heating the gel, decreases migrating of of DNA and distorts DNA bands

41
Q

The common technique to visualize the colorless nucleic acid is with use of….

A

Dyes

42
Q

SYBR safe DNA Gel Stain is a highly sensitive stain for visualization of….

A

Nucleotide strands in agarose or acrylamide gels

43
Q

SYBR safe binds with____ to form a complex that fluoresces under UV light

A

Double stranded DNA

44
Q

SYBR safe can also bind to….

A

Single stranded nucleotide strands (RNA)

45
Q

The size of DNA fragments in a sample is estimated by….

A

Comparing their sizes with the DNA marker

46
Q

_____ DNA digested with ____ enzyme is often used as a DNA marker for sizing and to approximate quantification of DNA

A

Lambda DNA digested with Hind III enzyme

47
Q

When DNA concentration is low, the smaller bands are ______

A

Invisible

48
Q

DNA is a ___ ___ but can fold and coil itself into more complex shapes

A

Double helix

49
Q

When DNA is cut upon digestion with enzymes, what happens to it?

A

It unfolds and becomes longer in size

50
Q

DNA may become ___ during the extraction process and create _____ in the ___ DNA

A

DNA may become NICKED during the extraction process and create NICKS in the CIRCULAR DNA

Biomedical Lab (10 decks)

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