lecture 11 - isolation of DNA and genes

Question 1

steps of DNA isolation

Answer 1

cell lysis
DNA purification from the cell extract
concentrate DNA
measurement of DNA purity and concentration

Question 2

what dont we want in our DNA sample

Answer 2

protein, ribosomes, mtDNA, lipid, plasmids

Question 3

cell lysis - biological

Answer 3

use enzymes to disrupt the cell membrane

Question 4

for plants use

Answer 4

cellulase

Question 5

for bacteria use

Answer 5

lysozyme

Question 6

for eukaryotic cells use

Answer 6

sappanin

Question 7

cell lysis - physical methods

Answer 7

osmosic pressure: excess water moves into the cell when cells are placed in hypotonic solution (low concentration of solutes) the cell bursts

Question 8

cell lysis - physical methods

Answer 8

freeze thaw, repeated cycles of freezing and thawing ruptures cell membranes through ice crystal formation

Question 9

cell lysis - mechanical methods

Answer 9

grinding:
pestle and mortar
bead mill
vortex

Question 10

DNA purification by phenol-chloroform extraction

Answer 10

lysed cells or tissues are mixed with equal volumes of phenol chloroform mixture
centrifugation = two distinct phases as the phenol:chloroform mixture does not mix with water
DNA concentration- 0.3M sodium acetate and 2.5 volumes ethanol can be used to precipitate DNA from salt and sugar to concentrate it

Question 11

DNA purification using commercial kits

Answer 11

column contains a silica membrane that binds DNA in the presence of a high concentration of salt
impurities such as salts are washed away and then a low salt buffer such as water or 10mM tris-Cl is used to release the DNA from the membrane and collect it
the kits are less hazardous, less time consuming and results are purer DNA than phenol chloroform extraction
chloroform - early anaesthetic
phenol - serious chemical burns

Question 12

measuring quantity and quality of DNA

Answer 12

UV absorbance
flourescence dyes
agarose gel electrophoresis
capillary electrophoresis
diphenylamine method
readings above 1.8 for pure DNA

Question 13

restriction endonucleases

Answer 13

enzymes produced by bacteria to protect against viral DNA infection

Question 14

how do they cut

Answer 14

make one cut in each of the sugar phosphate backbones of the double helix

Question 15

types of RE

Answer 15

some are blunt ends and some are sticky ends
recognition sites for RE are often palindromes

Question 16

multiple cloning site

Answer 16

allows for the insertion of foreign DNA without disrupting the rest of the plasmid