LAB - Electrophoresis

Question 1

the migration of charged solutes or particles within a liquid medium under the influence of an electrical field

Answer 1

ELECTROPHORESIS

Question 2

At pH 8.3, DNA is _______ charged and migrates from the ________ to the ______

Answer 2

negatively
cathode
anode

Question 3

Electroendosmosis

Answer 3

water moving in the opposite direction of desired migration that may affect migration
- caused by static, stationary neg charged acidic groups in the gel

Question 4

to determine size of DNA fragments

Answer 4

gel electrophoresis

Question 5

a technique used to resolve nucleic acid fragments based on their mass to charge ratio (m/z)

Answer 5

Electrophoresis

Question 6

in general, DNa and RNA migration through a support media such as agarose is based on this

Answer 6

size (they have the same mass to charge ratio or 1:1 ratio of charge to nucleotide mass so based on length of fragment)

Question 7

Factors influencing electrophoresis

Answer 7

• composition of agarose gel
• molecular size and conformation of the DNA
• applied voltage
• direction of the electrical field
• composition of the electrophoresis buffer
• presence of intercalating dyes

Question 8

agarose

Answer 8

linear polysaccharide polymer of agarobiose derived from seaweed

Question 9

T or F. electrophoretic mobility of DNA is linearly related to the concentration of agarose gel

Answer 9

T, the greater the percentage of agarose, the smaller the linear DNA that can be resolved

Question 10

T or F. the migration rate of linear DNA is inversely proportional to the voltage applied

Answer 10

F! proportional!
- BUT as the voltage is increased, migration rate of DNA fragments greater than 2kb is disproportionally enhanced compared to smaller molecules

Question 11

electrophoresis buffers

Answer 11

• weak acid and conjugate base
• pH maintained at a constant
• protects samples
• buffers are used to prepare gel and to run
• DNA buffers
  > Tris borate EDTA (TBE)
  > Tris Acetate (TAE)

Question 12

the pH of the electrophoresis buffer determines the _____ on the nucleic acid

Answer 12

charge

Question 13

EDTA purpose

Answer 13

chelates monovalent and divalent ions which are common cofactors for DNAse and RNAse

Question 14

components of the buffer also carry the _______ through the electrophoresis system. as the ionic strength of the buffer increases, migration of charged analytes in the gel _______. this has an ______ effect on resolution

Answer 14

current
decreases
adverse

Question 15

as the ionic strength of buffer increases, the amount of heat generated ________. large amounts of heat cause water to evaporate further increasing _____ strength of the buffer

Answer 15

increases

ionic

Question 16

a fluorescent dye which is used to visualize DNA in a gel

Answer 16

eithidium bromide

Question 17

fluorophore of ethidium bromide is excited at ___ m and is observed as ___ light

Answer 17

550

red

Question 18

T or F. the presence of ethidium bromide during electrophoresis decreases the migration rate of the DNA in agarose gels by up to 15%

Answer 18

T

Question 19

gel loading dyes functions

Answer 19

• to add density to sample
• to aid in visualizing while loading gel
• to monitor progress of electrophoresis

Question 20

loading dyes contain any of these to increase the density of the sample

Answer 20

glycerol
Ficoll
sucrose

Question 21

loading dyes for increased visualization

Answer 21

Xylene Cyanole
Bromophenol Blue
Orange G

Question 22

DNA visulaization dyes

Answer 22

• ethidium bromide = intercalating fluorescent nucleic acid dye; major groove of DNA
• SyBr Green = cyanine dye that has enhanced fluorescence upon binding to nucleic acids; minor groove; different excitation wavelength; 25-100x more sensitive
• Gel Red = intercalating fluorescent nucleic acid dye