Chapter 4 Flashcards
What do most restriction enzymes come with to provide optimal salts and pH?
A concentrated reaction buffer.
What additive do some restriction enzymes require?
Bovine serum albumin (BSA).
What is it called when two restriction enzymes digest DNA in the same tube?
A double digest.
At what temperature should many enzymes be stored?
–20°C.
Why are many enzymes supplied in glycerol?
To prevent freezing.
Why should repeated freeze-thawing of enzymes be avoided?
It reduces enzyme activity.
What is the main characteristic of freeze-dried enzymes?
They are designed for a single use but can be rehydrated for future use.
Why must rehydrated enzymes be further diluted before use?
To reduce the glycerol concentration to less than 5% in the final reaction.
How should enzymes be handled in the lab?
Kept on ice or at 4°C and returned to the freezer quickly.
Why should the freezer be open only briefly when retrieving enzymes?
To prevent temperature fluctuations.
What do Cas genes encode?
Enzymes that cut DNA in specific places.
How does a bacterium capture viral DNA?
It inserts viral DNA into a CRISPR sequence as a spacer.
What enzymes carry out the cut and capture in CRISPR?
Cas1 and Cas2.
What happens in the second phase of CRISPR defense?
Spacers are transcribed and bind to Cas9 to form a ‘search and destroy’ complex.
Why might CRISPR-modified plants face lighter regulations?
Because CRISPR can modify genes without introducing new genes.
What applications have scientists explored using CRISPR-Cas9 in yeast?
Producing lipids and polymers for biofuels, adhesives, and fragrances.
How has CRISPR been used in gene therapy research?
It has treated adult rats engineered to have genetic blindness.
How are agarose gels made?
By mixing agarose powder with 1x electrophoresis buffer and heating to melt the agarose.
At what temperature is molten agarose cooled before pouring?
Around 55°C.
What agarose percentage is used for separating fragments larger than 1 kb?
0.7–1%.
What type of agarose is used for DNA fragment recovery?
Low melting-point agarose.
What was originally used as DNA size standards?
Bacteriophage or plasmid DNA cut with restriction enzymes.
What is an example of a DNA standard using bacteriophage DNA?
Lambda phage DNA cut with HindIII.
How is the quantity of DNA in a band estimated?
By the intensity or thickness of the band.
What is a safety feature of the electrophoresis chamber?
A lid that prevents removal without breaking the current.
What feature do most power supplies for electrophoresis have?
A timer that turns off power when the run is complete.
What type of power supply does agarose gel electrophoresis require?
A standard power supply with mid-range voltage and current.
What is included in sample loading buffer to increase sample density?
Glycerol or Ficoll.
What color is the cathode (-) in electrophoresis?
Black.
What voltage is typically used for running agarose gels?
100 V for 30 minutes to 1 hour.
What is an example of a positively charged DNA stain?
Methylene blue.
When must positively charged stains be applied?
After the gel has been run.
What is a commonly used fluorescent DNA stain?
Ethidium bromide.
How is fluorescently stained DNA visualized?
Using UV or blue light.
Why is ethidium bromide hazardous?
It requires UV light, which is harmful and requires protective shielding.
What are two safer alternatives to ethidium bromide?
SYBR® Safe DNA stain and UView 6x loading dye.
How can DNA fragments stained with SYBR® Safe be viewed?
With a blue light source and an orange filter.
What system is required for viewing fluorescent DNA stains?
A gel documentation system with a transilluminator and camera.
How can images of positively charged stains be captured?
Using a white-light transilluminator.
Fragments <1 kb are usually separated with a _____ percentage of agarose
higher
High-strength agarose is available for separating
very large DNA fragments.
More recently, DNA size standards have been made from _______ that forms a regular “ladder” of DNA with precisely defined size intervals.
engineered DNA
When a bacterium or archaeon is infected by a virus,
the microbe captures some of the viral DNA and inserts it into a CRISPR sequence as a spacer
Researchers are looking to CRISPR as a technique for
editing out genetic defects
Specialty agaroses are available for separating
very small fragments
There are many different types of power supplies, most of which run How many electrophoresis chambers at a time?
2-4
DNA standards with more focused ranges are useful when
the sizes of the expected fragments are known and lie within the range of the ladder.
Most power supplies allow either the voltage or the current to be set by the —-
user
Western blotting requires a
power supply that can run at high current,
while sequencing gels require
a power supply with very high voltage.
—– is still the most common DNA stain used in research and industry
Ethidium bromide
UV light can also damage the — that is being viewed.
DNA
If fluorescent DNA stains are used, ————- is required to view and record the gel results
a gel documentation system consisting of a transilluminator, blue light (see Figure 4.20), and an image capturing system such as a camera
