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Q: What is the buffer composition for each endonucleases?

Why is it
unique for each endonuclease? What is the use of it?
A: Different restriction enzymes are optimally active in buffers with different salt
concentrations and may prefer different cations (usually sodium or potassium)
and anions (chloride or acetate). All digest buffers have DTT (dithiothreitol), which
reduces disulfides, preventing cysteines on different proteins from crosslinking
and disrupting their structure and activity. All have a source of magnesium ions
that, because of their size and positive charge, help stabilize the negatively
charged DNA phosphate backbone when the protein binds DNA. All have Tris, a
common buffering agent that works well at physiological pH ranges. This leaves
only the salt concentration and composition to tweak. In all cases, a major
function of the buffer is to maintain pH of the reaction (usually at 8.0).
Restriction Enzyme

BamHI
EcoRI
HindIII
PstI
ScaI
SaII
SapI
StyI

pH
(at
37oC)
7.5
7.5
7.5
7.5
7.4
7.9
7.4
8.5

Composition
TrisMgCl NaCl
HCl
(mM)
2
(mM) (mM)
6
6
100
90
10
50
6
6
100
90
10
50
10
10
6
6
150
10
300
10
10
100

KCl
(mM)
150
-

DTT
(mM
)
1
1
1
1
1

Q: Why should DNA be added last?


A:
Q: What is the composition of TAE buffer? What is the use of it?
A: Tris base, Acetic Acid, EDTA. In molecular biology it is used in
agarose electrophoresis typically for the separation of nucleic acids such
as DNA and RNA. It is made up of Tris-acetate buffer, usually at pH 8.0,
and EDTA, which sequesters divalent cations. TAE has a lower buffer capacity
than TBE and can easily become exhausted, but linear, double stranded DNA
runs faster in TAE.

Q: Why should there be a negative control?


A: The negative control is the standard of comparison, as it bears no DNA. The
negative control should NOT show the desired outcome.

Q: Why should we incubate the mixture for 1 hour at 37OC in a block


heater?
A: Most restriction enzymes cut best at 37C, but there are many exceptions.
Enzymes isolated from thermophilic bacteria cut best at temperatures ranging
from 50 to 65C. Some other enzymes have a very short half life at 37C and its
recommended that they be incubated at 25C.

Q: What is a gel loading dye? What is the composition of it? What is it used
for? Why should it be loaded separately in 1% agarose gel?
A: Gel loading dye is a dye to assess how "fast" your gel is running and a reagent
to render your samples denser than the running buffer (so that the samples sink
in the well). It contains 4g sucrose, 25 mg of bromophenol blue or xylene cyanol,
and 10mL of dH2O.

Q: What is 1% agarose gel? What is its composition? What is it used for?


What does it do?
A: Agarose gel is a substance that is used for gel electrophoresis and size
exclusion chromatography, which are methods of sorting large molecules by their
size and electrical charge. These processes use agarose to separate and
analyze proteins and DNA. In order to make 1% agarose gel, were going to need
1g agarose in 100mL TAE buffer.

Q: What is dna ladder? What is the composition of it? What is it used for?
Why only 50bp?
A: The substance known as DNA ladder, or just plain ladder, is a solution of
DNA molecules of different known lengths. The ladder allows you to estimate the
size of the unknown fragment by comparing it to the closest band in the ladder
lane. The steps, or rungs, of the DNA ladder are made up of two bases joined

together with either two or three weak hydrogen bonds. The rails of the ladder are
made up of alternating sugar and phosphate molecules. The rungs are
comprised of two bases between sugar molecules.
Q: What is electrophoresis? What does it do? Why do it? Why at 100v
250mA 50w?
A: Electrophoresis is a technique used to separate and sometimes purify
macromolecules - especially proteins and nucleic acids - that differ in size,
charge or conformation. It is fast, inexpensive, reliable.

Q: What is submarine gel electrophoresis system?


A:
Q: Why should TAE buffer cover up to maximum mark?
A: The TAE buffer fills the electrophoresis chamber and covers the gel, allowing
the electricity to conduct evenly through the gel. If the gel and buffer conduct
electricity too well, the gel and buffer will get hot. If this happens, our gel can
melt, and our DNA will denature. If the gel and buffer do not conduct electricity
well enough, our DNA will take too long to migrate through the gel, if it migrates
at all.
Q: Why should the anode be on the side of the well?
A: DNA is negatively charged at a neutral pH (which is something else our TAE
buffer helps us achieve), so if we expose the end of the gel nearest the DNA to a
negative charge and the end of the gel farthest from the DNA to a positive
charge, the DNA will migrate away from Gel Electrophoresis, BIO 2420 1 SKL12/01 the negative charge and toward the other end of the gel.
Q: Why ethidium bromide? What is it? What does it do? What is it used for.
Why shake for 15 minutes?
A: Ethidium bromide is a fluorescent dye used for staining nucleic acids. This
fluorescent dye intercalates between bases of DNA and RNA. It is often
incorporated into the gel so that staining occurs during electrophoresis, but the
gel can also be stained after electrophoresis by soaking in a dilute solution of
ethidium bromide. To visualize DNA or RNA, the gel is placed on a ultraviolet

transilluminator. Shaking is done in order to better see the streaks as it will


provide better staining.