LabPractical

Question 1

CTAB

Answer 1

Cetyltrimethyl Ammonium Bromide

Denatures proteins and solubilizes lipid membranes

Question 2

Proteinase K

Answer 2

Enzyme that breaks down proteins

Hydrolyzes peptide bonds

Highly active at pH 7.5-12 and 20-60 C

Question 3

Chloroform isoamyl alcohol

Answer 3

Separates nucleic acids from other cellular substances

Results in diphasic mixture: lower organic layer and upper aqueous layer containing DNA

Question 4

Ethanol

Answer 4

Promotes DNA aggregation leading to a pellet

Question 5

DNA degradation

Answer 5

Heat
Physical shearing
Repeated freezing/thawing
Room temp

Question 6

Proper DNA storage

Answer 6

2-8 degrees Celsius for one year

-20 degrees Celsius for 7 years

-80 degrees Celsius for more than 7 years

Slightly basic conditions

Question 7

Cause of DNA precipitation in ethanol

Answer 7

DNA is negatively charged due to phosphate group on backbone

Bc of polarity DNA is hydrophilic

Tries to minimize surface area exposed to nonpolar solvent

Question 8

Interpreting DNA on gel

Answer 8

Based on molecular weights

Smaller fragments travel further

Question 9

Equation to make gel

Answer 9

2.5% 30 ml agarose
.025 x 30 = .75 g

1% 30 ml agarose
.01 x 30= .3 g

Question 10

Steps to make gel

Answer 10

Agarose
Buffer
Microwave1 minute or until clear
Agitate by hand
Add gel red
Let cool
Pour into casting tray
Immediately rinse flask
Hardens in about 20 minutes

Question 11

GoTaq

Answer 11

Thermos aquaticus enzyme
DNTPs
Buffer
MgCl2

Question 12

Different temp cycles of PCR

Answer 12

1. Denatures template
2. Annealing of primers
3. DNR polymerase bind to template and synthesize PCR product

Question 13

How often restriction enzyme will cut

Answer 13

4^n

CC WGG
GGW CC

Once every 4^5 base pairs