lab techniques Flashcards
gel electrophoresis separates what
macromolecules (proteins, DNA, RNA)
what is used for proteins/small molecules in gel electrophoresis
polyacrylamide
what is used for larger molecules in gel electrophoresis
agarose
negatively charged molecules travel toward
anode at bottom
in gel electrophoresis
larger molecules move…
SLOWER in gel electrophoresis
what dye is used in gel electrophoresis
coomassie blue stain for visualization
native-PAGE is a method where
polyacrylamide gel electrophoresis method for proteins is utilized with NON-DENATURING conditions
proteins keep their native charge and structure so that…
they are separated based on charge and size
in native-PAGE
in SDS-page, a polyacrylamide gel electrophoresis method is used for
proteins using DENATURING conditions
sodium dodecyl sulfate denature the…
proteins and give them a uniform charge
part of SDS-PAGE
proteins separate solely on mass and then you can…
estimate the protein’s molecular mass
SDS-PAGE
reducing SDS-PAGE is the same as
SDS-PAGE but adds a reducing agent
reducing agent in reducing SDS-PAGE will reduce
disulfide bridges and result in completely denatured protein
isoelectric focusing is where a gel electrophoresis method is used to
separate proteins on the basis of relative contents of acidic/basic residues
gel has pH gradient and proteins will migrate through gel until….
they reach pH that matches isoelectric point
at the pI, the protein has a neutral charge so
it will no longer be attracted to the anode and it will stop migrating
part of isoelectric focusing
southern blotting
detection of a specific DNA sequence in a sample
northern blotting is the detection of
specific RNA sequences in a sample
western blotting is the detection of a
specific protein in a sample
sanger DNA sequencing uses what method
chain termination method
chain termination method uses
dideoxy nucleotides
ddNTP lacks a
hydroxyl group on 3’ carbon on sugar ring
no more nucleotides can be added to the chain w/ the 3’ hydroxyl group missing
chain ends with the
ddNTP that is marked with a particular color of dye depending on base carried
polymerase chain reaction (PCR) is used to make
many copies of a specific DNA region in vitro
key ingredients of PCR =
Taq polymerase
primers
template DNA
nucleotides (DNA building blocks)
ingredients are assembled in a tube along with cofactors needed by the enzyme and put through…..
repeated cycles of heating/cooling that allow DNA to be synthesized
primer must have high…
GC content and either a G or C at each end
enzyme used in PCR
taq polymerase
taq polymerase is named after the
heat-tolerant bacterium from which it is isolated
very heat-stable and most active around 70 C
steps of PCR
1) denaturation (96 C)
2) annealing (55 - 65 C)
3) extension (72 C)
PCR’s cycle is repeated until
you have enough DNA
chromatography separates
two or more molecules from a mixture
stationary phase is
typically polar
polar molecules elute
SLOWER
mobile phase is
typically nonpolar
nonpolar molecules elute
faster
liquid chromatography utilizes
silica in stationary phase
toluene/nonpolar liquid used as mobile phase
HPLC is a type of liquid chromatography that uses high pressure to pass…
the solvent phase through a more finely-group stationary phase
increases interactions b/w molecules and stationary phase
high-performance liquid chromatography (HPLC) has
higher resolving power
gas chromatography vaporizes liquid before
separation
molecules are separated based on
polarity and boiling point
stationary phase in gas chromatography is a
thin layer of material applied in column’s inside
the polarity of the stationary phase matches that…
of the solute in gas chromatography
mobile phase of gas chromatography is
an inert gas
size-exclusion chromatography separates molecules by
size rather than polarity
smaller molecules enter the porous gel beads, allowing them to
elute later
larger molecules elute faster because
they do not fit in the pores and will not be slowed down
ion-exchange chromatography separates
proteins by net charge
column is filled with
charged beads either positive or negative
part of ion exchange chromatography
cation exchange uses
negative beads
negative proteins elute first
anion exchange uses
positive beads
positive proteins elute first
affinity chromatography separates proteins based on
their affinity for a specific ligand
beads are bound to a specific ligand and proteins with a…
high affinity for that ligand will bind to the beads
affinity chromatography
proteins with a low affinity for the
ligand with elute first in affinity chromatography
thin-layer chromatography is a
sheet coated in polar silica gel
molecules are spotted at the
bottom of the sheet
thin-layer chromatography
sheet is placed in a nonpolar liquid and the mobile phase…
travels up the plate using capillary action
nonpolar molecules have the highest
Rx value
retention factor (Rf) =
distance spot moved / distance solvent front moved
central dogma
DNA –> transcription –> RNA –> translation –> PROTEIN
