Lab 9

Question 1

What is gel electrophoresis?

Answer 1

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

Question 2

Separation in gel electrophoresis depends on….

Answer 2

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

Question 3

Gel matrix contains…

Answer 3

Pores of a uniform size through which molecules can pass through

Question 4

Which makes larger pores - agarose or polyacrylamide?

Answer 4

Agarose

Question 5

What determines the size of the pores in gel electrophoresis?

Answer 5

Gel concentration

Question 6

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

Answer 6

Smaller

Question 7

What molecules are separated using agarose gel? Why?

Answer 7

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

Question 8

Polyacrylamide is used to separate____. Why?

Answer 8

Proteins because they are generally much smaller than DNA and RNA

Question 9

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

Answer 9

The size of the molecules

Question 10

Which migrate faster-large or small molecules?

Answer 10

Small

Question 11

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

Answer 11

Supercoiled

Question 12

When coiled, DNA is very ___ and ____

Answer 12

Small and compact

Question 13

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

Answer 13

Coiled will move faster because its very small and compact

Question 14

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

Answer 14

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

Question 15

How is the agarose gel prepared?

Answer 15

By heating the agarose powder in the buffer

Question 16

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

Answer 16

SPRINKLE, ROOM TEMP, 1 MIN

Question 17

Why is it best to follow this procedure?

Answer 17

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

Question 18

The hot liquid is poured into a ——-

Answer 18

Casting tray containing a comb

Question 19

The comb is removed from the casting tray when…

Answer 19

The gel solidifies to leave wells in the gels

Question 20

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

Answer 20

Mixed with a DNA LOADING BUFFER and loaded into WELLS

Question 21

What does the DNA loading buffer contain?

Answer 21

It is a concentrate containing a tracking dye and glycerol

Question 22

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

Answer 22

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

Question 23

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

Answer 23

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

Question 24

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

Answer 24

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

Question 25

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

Answer 25

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

Question 26

What will result in sharper bands?

Answer 26

Loading DNA in the smallest volume possible

Question 27

What do molecules need to move through the gel pores?

Answer 27

Electric current

Question 28

The higher the voltage, the ____ DNA moves

Answer 28

Faster

Question 29

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

Answer 29

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

Question 30

____ affects the migration of molecules through an electric field

Answer 30

Charge

Question 31

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

Answer 31

Cathode (negative electrode)

Question 32

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

Answer 32

The anode (positive electrode)

Question 33

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

Answer 33

NO

Question 34

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

Answer 34

A similar charge

Question 35

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

Answer 35

Because it is not a good conductor of electricity

Question 36

Why is a running buffer containing electrolytes used?

Answer 36

To ensure the conductance of electricity through the gel

Question 37

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

Answer 37

Negative charge because they both contain ionizable phosphate groups

Question 38

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

Answer 38

Because it provides better resolution for fragments >4kb

Question 39

The gel is covered ____-____mm with the buffer

Answer 39

2-3mm

Question 40

Too much buffer results in….

Answer 40

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

Question 41

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

Answer 41

Dyes

Question 42

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

Answer 42

Nucleotide strands in agarose or acrylamide gels

Question 43

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

Answer 43

Double stranded DNA

Question 44

SYBR safe can also bind to….

Answer 44

Single stranded nucleotide strands (RNA)

Question 45

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

Answer 45

Comparing their sizes with the DNA marker

Question 46

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

Answer 46

Lambda DNA digested with Hind III enzyme

Question 47

When DNA concentration is low, the smaller bands are ______

Answer 47

Invisible

Question 48

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

Answer 48

Double helix

Question 49

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

Answer 49

It unfolds and becomes longer in size

Question 50

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

Answer 50

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