Lab Techniques Flashcards
Gel Electrophoresis
Separates macromolecules (proteins, DNA, or RNA). For proteins and small molecules
the gel is polyacrylamide. For larger molecules (>500 bp), the gel is agarose.
Negatively charged molecules travel toward the anode at the bottom. Large
molecules will move SLOWER. Coomassie Blue stain can be used for visualization.
Native-PAGE
A polyacrylamide gel electrophoresis method for proteins using
NON-DENATURING conditions. Proteins keep their native charge
and structure so they are separated based on charge and size.
SDS-PAGE
A polyacrylamide gel electrophoresis method for proteins using
DENATURING conditions. Sodium Dodecyl Sulfate denatures the
proteins and gives the proteins a uniform charge. This allows
them to be separated solely on mass, thus, you can estimate the
protein’s molecular mass.
Reducing SDSPAGE
Exactly the same as SDS-PAGE, but with the addition of a
reducing agent, b-mercaptoethanol, which will reduce the
disulfide bridges and result in a completely denatured protein.
Isoelectric
Focusing
A gel electrophoresis method that separates proteins on the
basis of their relative contents of acidic and basic residues. The
gel has a pH gradient and the proteins will migrate through the
gel until they reach the pH that matches their 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.
Southern Blotting
Detection of a specific DNA sequence in a sample
Northern Blotting
Detection of a specific RNA sequence in a sample
Western Blotting
Detection of a specific PROTEIN in a sample
Chromatography
Separates two or more molecules from a mixture
Stationary Phase
Typically polar. Polar molecules elute slower
Mobile Phase
Typically nonpolar. Nonpolar molecules elute faster
Liquid Chromatography
Silica is used as the stationary phase while toluene or
another nonpolar liquid is used as the mobile phase.
High-Performance Liquid
Chromatography
HPLC is a type of liquid chromatography that uses high
pressure to pass the solvent phase through a more finelyground
stationary phase which increases the interactions
between the moelcuels and the stationary phase. This
gives HPLC higher resolving power.
Gas Chromatography
Vaporizes the liquid before separation. Molecules are
separated based on polarity and boiling point. The
stationary phase is a thin layer of material applied to the
inside of the column. Typically the polarity of the
stationary phase matches that of the solute. The mobile
phase is an inert gas.
Gel-Filtration
Chromatography:
(Size-exclusion
Separates molecules by size rather than polarity. Smaller
molecules enter the porous gel beads allowing them to
elute later. Larger molecules will elute faster because
they do not fit in the pores and will not be slowed down.
Ion Exchange
Chromatography
Separates proteins by their net charge. The column is
filled with charged beads, either POS or NEG.
Ion Exchange
Chromatography:
Cation Exchange
NEG beads used, NEG proteins elute 1st.
Ion Exchange
Chromatography:
Anion Exchange
POS beads used, POS proteins elute 1st
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.
Proteins with a low affinity for the ligand will elute first.
Thin-Layer
Chromatography
Sheet coated in polar silica gel. Molecules are spotted on
the bottom of the sheet. Sheet is placed in a nonpolar
liquid. Mobile phase travels up the plate using capillary
action. Nonpolar molecules have the highest Rf value.
PCR
Used to make many copies of a specific DNA region in vitro. The key ingredients of
PCR are Taq polymerase, primers, template DNA, and nucleotides (DNA building
blocks). The ingredients are assembled in a tube, along with cofactors needed by the
enzyme, and are put through repeated cycles of heating and cooling that allow DNA
to be synthesized.
PRC - Primer
Must have high GC content and either a G or C at each end.
Example: 5’-GCATAGAAGCATTCCGC-3’
PCR - Taq Polymerase
The DNA polymerase typically used in PCR. Named after the
heat-tolerant bacterium from which it is isolated (Thermos
aquaticus). Very heat-stable and most active around 70°C.
Steps: 1. Denaturation (96°C)
2. Annealing (55 - 65°C)
3. Extension (72°C)
Cycle is repeated until you have enough DNA
