Ch. 4 - Nucleic Acid Extraction Methods

Question 1

What is the first step to nucleic acid extraction?

Answer 1

Lysing the cell

Question 2

What mixture is used to lyse the cell? What are its components and their function?

Answer 2

Alkaline + detergent

SDS - dissolves lipid component of cell membrane and denatures proteins
NaOH - enatures both chromosomal and plasmid DNA into ss

Question 3

Organic Isolation of DNA

Answer 3

Utilizes solubility differences among chromosomal DNA, plasmid, and proteins in alkaline buffers

1. Lysis with SDS and NaOH
2. Acidification with acetic acid and salt
3. Extraction with phenol and chloroform
4. DNA precipitation with EtOH or isopropanol

Question 4

Inorganic Isolation of DNA

Answer 4

Aka “salting out”

Lysis with Tris, EDTA, and SDS
Precipitate protein with sodium acetate
Precipitate DNA with isopropanol

Question 5

Organic Isolation of RNA

Answer 5

Lysis with SDS and high salt OR with GITC
Extract with phenol and chloroform
Precipitate with EtOH or isopropanol

Question 6

DEPC

Answer 6

diethyl pyrocarbonate

Inactivates RNases permanently by cross-linking them

Question 7

rRNA

Answer 7

most abundant RNA in all cells
Large (28S) and small (18S) subunits
Two bands on agarose gel

Question 8

mRNA

Answer 8

Second most abundant RNA in all cells

Faint band on agarose gel

Question 9

Total RNA

Answer 9

rRNA, mRNA, tRNA, and snRNA

Question 10

RNA is extracted with _______.

Answer 10

chloroform/phenol/isoamyl alcohol

Question 11

Phenol/chloroform function

Answer 11

Denatures proteins

Question 12

Isoamyl alcohol function

Answer 12

Prevents foaming

Question 13

After organic RNA extraction, in which phase is the RNA found?

Answer 13

Top phase in aqueous solution

Question 14

What is the function of the acid neutralization solute in the isolation of DNA?

Answer 14

Bring the pH to neutral

This causes the plasmid DNA to renature first and the large chromosomal DNA to aggregate

Question 15

SDS/lipid/protein precipitate traps tangled _______ DNA.

Answer 15

chromosomal

Question 16

Mini-prep

Answer 16

Solid-phase isolation method
Spin columns to fit into eppendorf tubes
Solid matrices to bind and wash DNA
Bind DNA effectively at HIGH SALT concentrations

Question 17

Mini-prep procedure

Answer 17

Alkaline solution - denature proteins and DNA
Acid neutralization solution - plasmids renature
Supernatant has plasmid DNA
Silica column
Elution

Question 18

Solid-phase Isolation of RNA

Answer 18

Lysis (supplied reagents) 
RNA adsorption (low pH) 
Wash RNA (supplied buffer) 
Elute RNA (low salt)

Question 19

DNA spectrophotometry

Answer 19

1 OD 260 = 50 micrograms/mL dsDNA (conc.)
Conc. * mL = micrograms DNA (yield)
OD 260/OD 320 = 1.6-2.0 (purity)

Question 20

RNA spectrophotometry

Answer 20

1 OD 260 = 40 micrograms/mL RNA (conc.)
Conc. * mL = micrograms RNA (yield)
OD 260/OD 320 = >1.6 (purity)