Chapter 4

Question 1

What do most restriction enzymes come with to provide optimal salts and pH?

Answer 1

A concentrated reaction buffer.

Question 2

What additive do some restriction enzymes require?

Answer 2

Bovine serum albumin (BSA).

Question 3

What is it called when two restriction enzymes digest DNA in the same tube?

Answer 3

A double digest.

Question 4

At what temperature should many enzymes be stored?

Answer 4

–20°C.

Question 5

Why are many enzymes supplied in glycerol?

Answer 5

To prevent freezing.

Question 6

Why should repeated freeze-thawing of enzymes be avoided?

Answer 6

It reduces enzyme activity.

Question 7

What is the main characteristic of freeze-dried enzymes?

Answer 7

They are designed for a single use but can be rehydrated for future use.

Question 8

Why must rehydrated enzymes be further diluted before use?

Answer 8

To reduce the glycerol concentration to less than 5% in the final reaction.

Question 9

How should enzymes be handled in the lab?

Answer 9

Kept on ice or at 4°C and returned to the freezer quickly.

Question 10

Why should the freezer be open only briefly when retrieving enzymes?

Answer 10

To prevent temperature fluctuations.

Question 11

What do Cas genes encode?

Answer 11

Enzymes that cut DNA in specific places.

Question 12

How does a bacterium capture viral DNA?

Answer 12

It inserts viral DNA into a CRISPR sequence as a spacer.

Question 13

What enzymes carry out the cut and capture in CRISPR?

Answer 13

Cas1 and Cas2.

Question 14

What happens in the second phase of CRISPR defense?

Answer 14

Spacers are transcribed and bind to Cas9 to form a ‘search and destroy’ complex.

Question 15

Why might CRISPR-modified plants face lighter regulations?

Answer 15

Because CRISPR can modify genes without introducing new genes.

Question 16

What applications have scientists explored using CRISPR-Cas9 in yeast?

Answer 16

Producing lipids and polymers for biofuels, adhesives, and fragrances.

Question 17

How has CRISPR been used in gene therapy research?

Answer 17

It has treated adult rats engineered to have genetic blindness.

Question 18

How are agarose gels made?

Answer 18

By mixing agarose powder with 1x electrophoresis buffer and heating to melt the agarose.

Question 19

At what temperature is molten agarose cooled before pouring?

Answer 19

Around 55°C.

Question 20

What agarose percentage is used for separating fragments larger than 1 kb?

Answer 20

0.7–1%.

Question 21

What type of agarose is used for DNA fragment recovery?

Answer 21

Low melting-point agarose.

Question 22

What was originally used as DNA size standards?

Answer 22

Bacteriophage or plasmid DNA cut with restriction enzymes.

Question 23

What is an example of a DNA standard using bacteriophage DNA?

Answer 23

Lambda phage DNA cut with HindIII.

Question 24

How is the quantity of DNA in a band estimated?

Answer 24

By the intensity or thickness of the band.

Question 25

What is a safety feature of the electrophoresis chamber?

Answer 25

A lid that prevents removal without breaking the current.

Question 26

What feature do most power supplies for electrophoresis have?

Answer 26

A timer that turns off power when the run is complete.

Question 27

What type of power supply does agarose gel electrophoresis require?

Answer 27

A standard power supply with mid-range voltage and current.

Question 28

What is included in sample loading buffer to increase sample density?

Answer 28

Glycerol or Ficoll.

Question 29

What color is the cathode (-) in electrophoresis?

Answer 29

Black.

Question 30

What voltage is typically used for running agarose gels?

Answer 30

100 V for 30 minutes to 1 hour.

Question 31

What is an example of a positively charged DNA stain?

Answer 31

Methylene blue.

Question 32

When must positively charged stains be applied?

Answer 32

After the gel has been run.

Question 33

What is a commonly used fluorescent DNA stain?

Answer 33

Ethidium bromide.

Question 34

How is fluorescently stained DNA visualized?

Answer 34

Using UV or blue light.

Question 35

Why is ethidium bromide hazardous?

Answer 35

It requires UV light, which is harmful and requires protective shielding.

Question 36

What are two safer alternatives to ethidium bromide?

Answer 36

SYBR® Safe DNA stain and UView 6x loading dye.

Question 37

How can DNA fragments stained with SYBR® Safe be viewed?

Answer 37

With a blue light source and an orange filter.

Question 38

What system is required for viewing fluorescent DNA stains?

Answer 38

A gel documentation system with a transilluminator and camera.

Question 39

How can images of positively charged stains be captured?

Answer 39

Using a white-light transilluminator.

Question 40

Fragments <1 kb are usually separated with a _____ percentage of agarose

Answer 40

higher

Question 41

High-strength agarose is available for separating

Answer 41

very large DNA fragments.

Question 42

More recently, DNA size standards have been made from _______ that forms a regular “ladder” of DNA with precisely defined size intervals.

Answer 42

engineered DNA

Question 43

When a bacterium or archaeon is infected by a virus,

Answer 43

the microbe captures some of the viral DNA and inserts it into a CRISPR sequence as a spacer

Question 44

Researchers are looking to CRISPR as a technique for

Answer 44

editing out genetic defects

Question 45

Specialty agaroses are available for separating

Answer 45

very small fragments

Question 46

There are many different types of power supplies, most of which run How many electrophoresis chambers at a time?

Answer 46

2-4

Question 47

DNA standards with more focused ranges are useful when

Answer 47

the sizes of the expected fragments are known and lie within the range of the ladder.

Question 48

Most power supplies allow either the voltage or the current to be set by the ----

Answer 48

user

Question 49

Western blotting requires a

Answer 49

power supply that can run at high current,

Question 50

while sequencing gels require

Answer 50

a power supply with very high voltage.

Question 51

----- is still the most common DNA stain used in research and industry

Answer 51

Ethidium bromide

Question 52

UV light can also damage the --- that is being viewed.

Answer 52

DNA

Question 53

If fluorescent DNA stains are used, ------------- is required to view and record the gel results

Answer 53

a gel documentation system consisting of a transilluminator, blue light (see Figure 4.20), and an image capturing system such as a camera