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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: 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 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.