Practical 3

Question 1

plasmid

Answer 1

autonomously replicating dsDNA molecules found in bacteria and yeast
can supercoil
can use as vectors to insert and carry genes
ie- can add Antibiotic resistant gene and grow on Antibiotic so only plasmids taking up gene can grow

Question 2

PCR requirements

Answer 2

amplifies ds DNA
need Taq polymerase
Primers
dNTPS
H20
magnesium and ions
heat

Question 3

alkaline lysis

Answer 3

use this to purify DNA
suspend cells in RNase and neutral buffer
add NaOH and KDS (detergent) - lyses bacteria and denatures proteins and dsDNA, plasmid DNA denatured but strands stay together
add potassium acetate - lower pH and plasmid DNA renatures
spin to get rid of precipitate
pass solution through silica colmn (DNA binds to column, other contaminants washed off)
elute plasmid in EB

Question 4

why may plasmid with no enzyme have many tracks

Answer 4

supercoiling occurs
higher band- nicked strand (less coiling)
even higher band (plasmids circulising and forming concatomers

Question 5

types of restriction enzyme cutting

Answer 5

assymetric and symmetric cutting

Question 6

southern blotting

Answer 6

run DNA gene in gel, probe membrane with RNA complementary and use flourescence

Question 7

northern blotting

Answer 7

run rna, use comp DNA to find RNA sequence

Question 8

bacterocidal

Answer 8

kills bacteria

Question 9

bacteriostatic

Answer 9

prevents bacteria replication

Question 10

why do bacteria not digest own DNA with REs

Answer 10

For every RE, produce enxyme that methylates at that site to prevent degradation