Lab5+6

Question 1

What is in Urea Buffer

Answer 1

Urea
NaCL
EDTA
Sarksoly (TENS)

Question 2

Urea function

Answer 2

cell lysis + prvents 2º metabolites from interfereing

Question 3

TRIS function

Answer 3

maintain pH + increase permeability

Question 4

EDTA

Answer 4

chealting agent + deactivates DNAse

Question 5

NACL

Answer 5

prevents DNA deactivation

Question 6

Phenol

Answer 6

unfolds proteins structure

Question 7

Choloform

Answer 7

seperartes organic + aq layer (keeps DNA in aq layer)

Question 8

Ammonium Acetate

Answer 8

precipitates DNA

Question 9

What are 6 requirements of an ideal primer design

Answer 9

18-24 bp long
40-60 GC content
start + end with 1-2 GC pairs
Melt temp 50º-60º with 5º between each other
not complimentary

Question 10

What does spectrophotometric analysis entail

Answer 10

• absorbance/transmission of light through liquid
  260: NA
  230: organic + proteins
  280: proteins
  1.8-2..0

Question 11

What are the phases of the basic PCR program

Answer 11

1. initial denaturation: 2 mins at 94-95 to denature dsDNA
2. final denaturation: 30s at 94 to make sure
3. anneal: f+r primers are stable to anneal to ssDNA AND polymerase stable to bind to primers
4. extension: 1 min at 72, taq is at optimal (70-75) , DNA polymerase can syntheise and elongate
5. final extension: 5 mins at 72ºC to fill in protdruing ends