DNA/Protein Isolation

Question 1

What is the preferred gel for DNA electrophoresis?

Answer 1

agarose gel

Question 2

What is the preferred gel for protein electrophoresis?

Answer 2

polyacrylamide gel

Question 3

Gel electrophoresis is a technique used to separate proteins AND DNA based on two characteristics. What are these characteristics?

Answer 3

1. size
2. charge

Question 4

In an electrolytic cell (gel electrophoresis), is a cathode the negatively or positively charged electrode? Anode?

Answer 4

• cathode: negatively charged (attracts cations)
• anode: positively charged (attracts anions)

Question 5

[Mnemonic]

RED CAT and an AN OX

(how to remember where reduction and oxidation occurs)

Answer 5

RED CAT and an AN OX

RED CAT: reduction occurs at the cathode

AN OX: oxidation occurs at the anode

Question 6

[Mnemonic]

LEO the lion goes GER!

(how to remember oxidation and reduction)

Answer 6

LEO the lion goes GER

• LEO: lose electrons oxidation
• GER: gain electrons reduction

Question 7

What are two kinds of electrochemical cells?

Answer 7

1. galvanic cell

(spontaneous because it provides its own power, i.e. a battery)

1. electrolytic cell

(nonspontaneous because it requires power from an outside source, i.e. gel electrophoresis)

Question 8

What is the function of the anionic detergent sodium dodecyl sulfate (SDS)?

Answer 8

Denatures proteins.

(Electrophoretic mobility in SDS PAGE depends mainly on size.)

Question 9

What is a zwitterion?

“or hybrid”

Answer 9

A molecule with both a positive AND negative charge, it is electrically neutral.

Question 10

What is isoelectric point (pI)?

Answer 10

The pH at which a protein or amino acid is electrically neutral.

Question 11

Explain what occurs during isoelectric focusing.

Answer 11

1. At some given pH a mixture of amino acids are placed in the center of a gel.
2. AA with a net positive charge will move towards the cathode. AA with a net negative charge will move towards the anode. AA with a net neutral charge will NOT move.
3. AA migrate to their pI.

Question 12

Isoelectric focusing separates proteins (or amino acids) based on what?

Answer 12

Based on their isoelectric point (pI).

Question 13

What is the stationary phase in ion exchange chromatography?

Answer 13

Beads with charges

The beads in a column have charges. Opposite charges attract and like charges repel and those components elute quickly through the column.

Question 14

In size exclusion chromatography which molecules elute first, the large or small?

Answer 14

Large

Question 15

In chromatography, if a component of a sample has high affinity for the stationary phase, how will it move?

Answer 15

It will barely move because it has a high affinity (more attraction) to the stationary phase.

Question 16

In chromatography, if a component of a sample has low affinity for the stationary phase, how will it move?

Answer 16

It will migrate quickly because it has a low affinity for the stationary phase, which means that is has high affinity (more attraction) to the mobile phase.

Question 17

What is the stationary phase in size-exclusion chromatography?

Answer 17

Beads with tiny pores of varying sizes

The tiny pores allow small compounds to enter which slows them down. Large compounds are too big and move around the beads and elute quickly through the column.

Question 18

For gel electrophoresis, which kind of molecules migrate through the gel quickly/__easily?

Answer 18

1. small
2. highly charged

Question 19

For gel electrophoresis, which kind of molecules migrate through the gel slowly?

Answer 19

1. large
2. electrically neutral

The larger the molecule, the more slowly it migrates.

Question 20

DNA polymerase in the human body does NOT work at high temperatures (heat), so a different polymerase must be used instead for PCR. Which polymerase can withstand the high temperatures and denature DNA strands in PCR?

Answer 20

Taq polymerase

(from Thermus aquaticus, a bacteria located in the hot springs of Yellowstone National Park)

Question 21

What does a Southern blot identify? Northern blot? Western blot?

Answer 21

• Southern blot: DNA
• Northern blot: RNA
• Western blot: proteins

Question 22

Which method is used to analyze proteins in their native states?

Answer 22

PAGE

(polyacrylamide gel electrophoresis)

(non-reducing/non-denaturing)

Question 23

What is the amount of time a compound spends in the stationary phase of chromatography called?

Answer 23

Retention time

Question 24

Which two methods are used in determining protein structure?

Answer 24

1. X-ray crystallography
2. nuclear magnetic resonance (NMR) spectroscopy

Question 25

What are the three main steps in polymerase chain reaction (PCR)?

Answer 25

1. denaturation (with heat)
2. reannealing
3. elongation

Question 26

What is the main purpose of polymerase chain reaction (PCR)?

Answer 26

Amplify DNA to produce millions of copies of a particular DNA sequence.

Question 27

What are the four main steps in a Southern Blot?

Answer 27

1. cleave DNA with restriction enzymes
2. perform DNA gel electrophoresis
3. transfer DNA from gel to another membrane (AKA blotting)
4. membrane is then probed with radioisotopes to locate the DNA sequence of interest