L6 Gel Electrophoresis, separation and detection

Question 1

the test must be able to detect very small amounts of target even in the presence of other molecules

Answer 1

Sensitivity

Question 2

the test yields a positive result for the target molecule only

Answer 2

Specificity

Question 3

the test must be able to run efficiently and inexpensively on a routine basis

Answer 3

Simplicitiy

Question 4

Lyze Cells
Enzymatic reactions
Chemical extractions and Purificiation

Answer 4

Isolating Nucleic Acids for Molecular Analysis

Question 5

Detergent SDS
Tween 20 etc..

Answer 5

Lyze cells

Question 6

Degrade Proteins
Lipids
Other Cellular Molecules

Answer 6

Enzymatic Reactions

Question 7

Organic and Inorganic

Answer 7

Chemical extraction and purification

Question 8

Phenol-chloroform-isoamyl alcohol

Answer 8

Organic extraction

Question 9

Salt precipitation
Adsorption to silica surfaces/matrix columns
Anion-exchange chromatography protocols

Answer 9

Inorganic Extraction

Question 10

OD260 = 1.0 ~50ug/ml of dsDNA or 40 ug/ml of RNA or ssDNA

Answer 10

Spectrophotometry
- Concentration abs at 260 nm (na bases)

Question 11

Ratio < 1.8 contamination with proteins or phenol

Answer 11

Purity (Spectrophotometry)

Question 12

Fluorometric or gel electrophoresis detection and quantification (EtBr, acridine orange, diamino-benzoic acid)

Answer 12

Fluorescent dyes

Question 13

• Restriction endonuclease enzyme digestion
• Gel electrophoresis and ETBr Stain
• Restriction Fragment Length Polymorphism (RFLP)

Answer 13

Direct dectection of NA

Question 14

• Found ONLY in microorganisms
• Function as homodimer; recognize symmetrical dsDNA (palindromes)

Answer 14

Restriction endonucleases

Question 15

• Palindrome reads the same in both directions
• Cutting genomic DNA with a RE results in many fragments of different sizes
• Sequences directly opposite one another on opposite strands of the dsDNA molecule

Answer 15

Restriction Enzymes Recognize Palindromes

Question 16

Example: BamH1 (5’ overhang)

Answer 16

Sequences directly opposite one another on opposite strands of the dsDNA molecule

Question 17

The negatively charged particles move toward the positive electrode while the positive charged particles move toward the negative electrode

Answer 17

Gel Electrophoresis

Question 18

Charge
Size
Shape

Answer 18

Molecules sort based on

Question 19

highly purified polysaccharide derived from agar (extracted from seaweed), long, sugar polymers held together by hydrogen and hydrophobic bonds.

Answer 19

Agarose