Week 5 - Electrophoresis

Question 1

Electrophoresis (general principle)

Answer 1

Charged particles (liq medium) migrate due to electrical influence
Separation based on charge and size
pH > pKa = neg charge
Neg molecules travel from CATHODE (B,-) -> ANODE (R,+)

Question 2

pH and pKa calculation

Answer 2

pH = -log[H+]
pKa = -logKa

Question 3

Agarose gel

Answer 3

[Low] = large pores 0.5%
[High] = small pores, increased gel strength, increased resolution 2%

Question 4

Electroendosmosis (EEO)

Answer 4

Charged groups (OH-) fixed negative charge on agarose gel. When electrical field is present, cations migrate towards the cathode (dragging water with them). Water movement towards the cathode inhibits the migration of negative particles (prot, DNA) towards anode)

Question 5

Buffers

Answer 5

TAE - Tris Acetate Ethylenediaminetetraacetuc Acid
TBE - Tris Borate Ethylenediaminetetraacetic Acid
SDS - Sodium Dodecylsulfate

Question 6

Benefit of Capillary Electrophoresis

Answer 6

Less voltage require, creating less heat
Smaller sample size
Faster run

Question 7

Ladder

Answer 7

Reference/standard with known bp size for comparison

Question 8

DNA Stain

Answer 8

Ethidium Bromide
SYBR Green
GEL red

Question 9

Problems/Errors

Answer 9

1. No migration - current connection, wrong buffer pH
2. Uneven banding, slanted - gel solidification, impurities
3. Band smearing - sample overload, thick gel, bubbles when pipetting
4. Sample denatured - heat
5. Loss of sample, spill from well - air bubbles
6. Excessive heat - increased ionic strength, decreased migration
7. Band runoff - too high voltage, too long run time

Question 10

Voltage

Answer 10

v = current x resistance

Question 11

Wick Flow

Answer 11

Movement of water from buffer res towards centre to gel or strip to replace water loss (via evaporation)

Question 12

Loading Dyes (Weight)

Answer 12

Glycerol, sucrose, glycol

1. visualization
2. density
3. track progress