Lab Techniques

Question 1

Gel Electrophoresis

Answer 1

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.

Question 2

Native-PAGE

Answer 2

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.

Question 3

SDS-PAGE

Answer 3

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.

Question 4

Reducing SDSPAGE

Answer 4

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.

Question 5

Isoelectric

Focusing

Answer 5

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.

Question 6

Southern Blotting

Answer 6

Detection of a specific DNA sequence in a sample

Question 7

Northern Blotting

Answer 7

Detection of a specific RNA sequence in a sample

Question 8

Western Blotting

Answer 8

Detection of a specific PROTEIN in a sample

Question 9

Chromatography

Answer 9

Separates two or more molecules from a mixture

Question 10

Stationary Phase

Answer 10

Typically polar. Polar molecules elute slower

Question 11

Mobile Phase

Answer 11

Typically nonpolar. Nonpolar molecules elute faster

Question 12

Liquid Chromatography

Answer 12

Silica is used as the stationary phase while toluene or

another nonpolar liquid is used as the mobile phase.

Question 13

High-Performance Liquid

Chromatography

Answer 13

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.

Question 14

Gas Chromatography

Answer 14

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.

Question 15

Gel-Filtration
Chromatography:
(Size-exclusion

Answer 15

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.

Question 16

Ion Exchange

Chromatography

Answer 16

Separates proteins by their net charge. The column is

filled with charged beads, either POS or NEG.

Question 17

Ion Exchange
Chromatography:
Cation Exchange

Answer 17

NEG beads used, NEG proteins elute 1st.

Question 18

Ion Exchange
Chromatography:
Anion Exchange

Answer 18

POS beads used, POS proteins elute 1st

Question 19

Affinity Chromatography

Answer 19

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.

Question 20

Thin-Layer

Chromatography

Answer 20

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.

Question 21

PCR

Answer 21

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.

Question 22

PRC - Primer

Answer 22

Must have high GC content and either a G or C at each end.

Example: 5’-GCATAGAAGCATTCCGC-3’

Question 23

PCR - Taq Polymerase

Answer 23

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