DNA

Question 1

ecoR1 methyltransferase

Answer 1

adds CH3 to second A of GAATTC sequence

Question 2

restriction endonuclease

Answer 2

cuts palindromic sequence if unmethylated

Question 3

type 1 restriction enzyme

Answer 3

multi enzyme subunit, cuts random sequence hundreds of base pairs away

Question 4

type II restriction enzyme

Answer 4

single protein, cut within that sequence

Question 5

type III restriction enzyme

Answer 5

multi enzyme subunit, random sequence cut 25 bp away

Question 6

strategies for making a map

Answer 6

• determine if it’s circular or linear
• make single digest maps, then combine them
• digests that give fewer bands are easier to map first
• compare single and double digest sizes to determine the identity of the bands
• keep track of all sizes/distances

Question 7

pulsed field gel electrophoresis

Answer 7

switches the run direction to counter reptation(fragments up to 1Mb can be resolved)(can identify strain specific patterns

Question 8

plasmid precipitation

Answer 8

salt added to supernatant
1-3 volumes of alcohol added to solution
chill on ice
spin in centrifuge to collect DNA
wash with 70% ethanol
Dry and resuspend in H20 or TE

Question 9

differential precipitation

Answer 9

using salts and alcohol

Question 10

solvent extraction

Answer 10

phenol/chloroform to remove contaminants

Question 11

plasmid precipitation common modifications

Answer 11

resuspend and lyse in one step
addition of lysozyme to lysis buffer for Gram (+)
remove contaminating proteins using phenol/chloroform extraction
resuspension of plasmid in buffer containing RNAase

Question 12

concept of PCR

Answer 12

using a heat stable enzyme and DNA building blocks we can make billions of copies of a DNA fragment from a single copy