Capillary Electrophoresis

Question 1

What role does buffer have in electrophoresis?

Answer 1

Supplies ions for the conduction of current
Dissolves polymer in CE systems
Stabilizes and solubilizes DNA

Question 2

Why is high quality formamide typically used as a diluent in CE of DNA fragments?

Answer 2

Low conductivity and DNA denaturation

Question 3

(True or False) When formamide is exposed to air it breaks down forming formic acid which can compete with the DNA fragments during injection.

Answer 3

True

Question 4

When an electric field is applied during ______, the DNA molecules will move away from the negatively charged ______ electrode towards the positively charged ______ electrode.

Answer 4

Electrophoresis
Cathode
Anode

Question 5

What method is used to add DNA amplicons to a capillary for electrophoresis?

Answer 5

Electrokinetic Injection

Question 6

(True or False) Separation media and/or buffers used in electrophoresis do not contain a denaturant because double-stranded DNA is desired.

Answer 6

False; single stranded DNA is desired

Question 7

What is added to each sample being injected on a CE system to determine the size of targeted DNA amplicons?

Answer 7

Internal Sizing Standard/ Internal Lane Standard

Question 8

(True or False) The fluorescent dyes that are attached to the amplicons being sized on a CE instrument fall within the visible region of the light spectrum.

Answer 8

True

Question 9

What is performed on a CE instrument to determine the relationship between the capillary and the camera?

Answer 9

Spatial Calibration

Question 10

(True or False) The fluoresce that is released from the dyes attached to the 3’ ends of the amplicons is detected by a charge coupled device camera.

Answer 10

False; 5’ ends

Question 11

Why is a spectral resolution needed when sizing DNA fragments with a CE instrument?

Answer 11

To differentiate between different fluorescent dyes

Question 12

(True or False) CE capillaries must not be allowed to dry out as it will cause clogging due to salt crystallization.

Answer 12

True

Question 13

How often are the capillaries filled with fresh polymer?

Answer 13

Before each injection

Question 14

What is contained in Performance Optimized Polymer-4 (POP 4) that keeps DNA denatured?

Answer 14

Urea

Question 15

What type of lasers are used on CE instruments?

Answer 15

Solid-state lasers

Question 16

(True or False) During electrophoresis, the DNA fragments are separated by size only since all DNA has equivalent mass to charge ratios.

Answer 16

True

Question 17

How many capillaries may be contained within a 3500 capillary array?

Answer 17

8 or 24 (24 at LSPCL)

Question 18

What length array is used most in fragment size analysis on a 3500 instrument?

Answer 18

36cm

Question 19

What is the minimum number of days that the anode and cathode buffer containers may be used for CE runs on a 3500?

Answer 19

14 days (manufacturer)
7 days (LSPCL)

Question 20

A laser is directed through the ______ window, this causes ______ of the dyes attached to the 5’ end of the amplicons and leads to the ______ of fluorescence.

Answer 20

Detection
Excitation
Emission

Question 21

What is the electrophoretic flow?

Answer 21

Electrophoretic flow is the movement of negatively charged DNA from the cathode to the anode.

Question 22

What is the electroosmotic flow?

Answer 22

Electroosmotic flow is the movement of the anode buffer to the cathode.

Question 23

What is electrophoresis?

Answer 23

The movement of charged particles through an electric field.

Question 24

Describe the inner capillary environment.

Answer 24

-DNA moves through the capillary with the electrophoretic flow from the cathode to the anode
-Linear, flexible polymer chains act as obstacles that must be navigated by the negatively charged DNA fragments on their way to the anode
-The speed of their movement through this matrix is based on the size of the DNA fragments
-The capillary glass/fused silica walls are negatively charged, and positive ions from the buffer solution line up on the capillary wall
-The electroosmotic flow is opposite of the electrophoretic flow and is the movement of the anode buffer toward the cathode. the polymer reduces the backward flow so DNA can still move toward the positive charge.

Question 25

How does the matrix affect the speed?

Answer 25

Larger particles move slower than smaller particles due to slowing movement through the matrix.

Question 26

What are the 3 requirements for STR typing in CE?

Answer 26

Spectral resolution, spatial resolution, DNA sizing precision

Question 27

During capillary electrophoresis, the data from the detector is measured in _______ and _______.

Answer 27

time and intensity