DNA Extraction

Question 1

method to purify DNA by using physical and/or chemical methods from a sample separating DNA from cell membranes, proteins, and other cellular components

Answer 1

DNA Extraction

Question 2

General Steps of DNA Extraction

Answer 2

Lysis > Precipitation > Purification (Washing and Elution)

Question 3

In this step, the nucleus and cell are broken first for the release of DNA

Answer 3

Lysis

Question 4

Plast cells possess tough cell walls, so ___ is significant.

Answer 4

Mechanical disruption

Question 5

For softer cell walls, ____ are used to free the cellular proteins and DNA

Answer 5

detergents and enzymes like proteinase K

Question 6

This step helps in the separation of DNA from the cellular debris

Answer 6

Precipitation

Question 7

During precipitation, their solubility in water ____

Answer 7

decreases

Question 8

This is the 3rd step where the purified DNA is redissolved in water or buffer for storage and handling with ease

Answer 8

Purification

Question 9

Detailed Steps of DNA Extraction

Answer 9

Spin down cells at 1,000 x g for 30 seconds > Ice-cold Soln I > Soln II > Ice-cold Soln III > Absolute Ethanold > Ice-cold 70% Isopropanol > TE Buffer

Question 10

Components of Ice-Cold Soln I

Answer 10

50mM glucose
25 mM Tris Cl
10 mM EDTA

Question 11

Glucose maintains the ____ so cells dont burst

Answer 11

osmotic pressure

Question 12

Tris HCI acts as a buffer to prevent _____ of closed, circular plasmid DNA

Answer 12

irreversible denaturation

Question 13

EDTA chelates divalent cations in the solution prevents DNases from _____ and helps ____

Answer 13

damaging plasmid; destabilizing cell wall

Question 14

NaOh helps to ___, bust more importantly disrupts ____ between the DNA bases

Answer 14

break down cell wall; hydrogen bonding (dsDNA and gDNA to ssDNA)

Question 15

SDS ___ the cells membrane and ____ most of the proteins in the cells

Answer 15

solubilizes; denatures

Question 16

Components of Solution II

Answer 16

0.2 N NaOh
1% SDS

Question 17

Components of Ice-cold Solution II

Answer 17

5 M potassium acetate
Glacial acetic acid

Question 18

the addition of ____ and ___ decreases the alkalinity of the mixture

Answer 18

potassium acetate and acetic acid

Question 19

___ helps to remove the remaining salts and SDS from the preparation and precipitates DNA

Answer 19

Absolute Ethanol

Question 20

70% Isopropanol

Answer 20

cleans DNA and removes other contaminants

Question 21

TE (Tris-EDTA) buffer is made up of

Answer 21

Tris, pH buffer, and EDTA

Question 22

is a metal chelating ion

Answer 22

EDTA

Question 23

TE buffer

Answer 23

used to wash and dissolve DNA

Question 24

Also used for DNA storage because of its ability to store DNA for a longer period of time without degrading it

Answer 24

TE Buffer

Question 25

How to Handle DNA

Answer 25

introduction of nucleases to DNA soln should be avoided Excessive vortexing/pipetting should be avoided Keep it cold Purified DNA should be stored at -20C or 70C under slightly basic conditions

Question 26

Storing Purified DNA in acidic conditions can cause

Answer 26

hydrolysis of DNA