Lab Techniques

Question 1

What is gel electrophoresis used for?

Answer 1

Macromolecules are separated within the gel as smaller molecules move toward the bottom.
Native = no denature
SDS = denatured protein
Reducing SDS = complete denature
Isoelectric = separation based on pH of residues

Question 2

SNoW DRoP

Answer 2

Southern Blotting = DNA

Question 3

What is western blotting?

Answer 3

A techinique that uses gel electrophoresis that uses DENATURED proteins that are then labeled with flourescent antibody then washed and exposed to excitation light and antibody is detected

Question 4

What are southern and northern blotting?

Answer 4

DNA (southern) or RNA (northern) are placed in polyacrylamide if less than 500 base pairs or agarose if more then 500 base pairs then radiolabeled 32p on molecule of interest then autoradiography is conducted and seen on a autoradiogram

Question 5

DNA Sequencing. Explain the method

Answer 5

DNA is denatured by NaOH then a radiolabeled complementary strand is added, all four ddNTPs are in small amounts for each (A, C, G, T), electrophoresis is conducted and autoradiography is used to identify strands.

Question 6

What is chromatography used for? And what are the different types.

Answer 6

Chromatography is used to separate or analyze a mixture of two or more molecules based on their properties.
Stationary phase = polar
Mobile phase = non polar
Reverse phase = properties of phase are switched

Question 7

Liquid chromatography

Answer 7

Silica is used in stationary and toluene or another non polar liquid is used as mobile phase

Question 8

High performance liquid chromatography

Answer 8

Usedhigh pressure

Question 9

Column chromatography

Answer 9

Using silica (polar) and solvent (no polar) the mixture is added. The polar silica makes part of the mixture move down the column slower and the non polar part of the mixture moves down faster.

Question 10

Gas chromatography (based on boiling points)

Answer 10

Noble (aka inert) gas is pushed through a coiled tube (which is the mobile phase) in which stationary liquid rests. The tube is heated in and the rate at which the compounds hit the detector is measured.

Question 11

Size exclusion chromatography

Answer 11

Separates molecules by size rather than polarity. Smaller molecules elute slower and larger ones elute faster.

Question 12

Ion exchange chromatography

Answer 12

Separates proteins by their net charge. Negative or positive beads are placed in a column and positive parts of mixture either attract or repel at different speeds.

Question 13

Thin layer chromatography

Answer 13

Plate that’s covered in silica gel, take a spotter and place a dot on the plate.

Question 14

Affinity chromatography (affinity for ligand)

Answer 14

Use of specific ligand in column and then the mixture is added to column, unused products elute first and target protein elites last.

Question 15

Simple distillation

Answer 15

Separates two molecules from a solution with greater than 25 degrees difference

Question 16

Fractional distillation

Answer 16

Separates molecules with LESS than 25 degree difference

Question 17

Vacuum distillation

Answer 17

Used when boiling points are high and they might chemically change

Question 18

Polymerase chain reaction (dna separation)

Answer 18

DNA is heated to 95 degrees

Question 19

Racemic mixtures and separation of enantiomers

Answer 19

Racemic mixtures are optically inactive. When they’re separated the mixture is resolved. A chiral agent is added that created diasteromers that can be then separated.

Question 20

What is extraction?

Answer 20

The act of separating a mixture by adding organic solvent and aqueous solvents then shaking the mixture to separate the two