Capillary Electrophoresis Flashcards
What role does buffer have in electrophoresis?
Supplies ions for the conduction of current
Dissolves polymer in CE systems
Stabilizes and solubilizes DNA
Why is high quality formamide typically used as a diluent in CE of DNA fragments?
Low conductivity and DNA denaturation
(True or False) When formamide is exposed to air it breaks down forming formic acid which can compete with the DNA fragments during injection.
True
When an electric field is applied during ______, the DNA molecules will move away from the negatively charged ______ electrode towards the positively charged ______ electrode.
Electrophoresis
Cathode
Anode
What method is used to add DNA amplicons to a capillary for electrophoresis?
Electrokinetic Injection
(True or False) Separation media and/or buffers used in electrophoresis do not contain a denaturant because double-stranded DNA is desired.
False; single stranded DNA is desired
What is added to each sample being injected on a CE system to determine the size of targeted DNA amplicons?
Internal Sizing Standard/ Internal Lane Standard
(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.
True
What is performed on a CE instrument to determine the relationship between the capillary and the camera?
Spatial Calibration
(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.
False; 5’ ends
Why is a spectral resolution needed when sizing DNA fragments with a CE instrument?
To differentiate between different fluorescent dyes
(True or False) CE capillaries must not be allowed to dry out as it will cause clogging due to salt crystallization.
True
How often are the capillaries filled with fresh polymer?
Before each injection
What is contained in Performance Optimized Polymer-4 (POP 4) that keeps DNA denatured?
Urea
What type of lasers are used on CE instruments?
Solid-state lasers
(True or False) During electrophoresis, the DNA fragments are separated by size only since all DNA has equivalent mass to charge ratios.
True
How many capillaries may be contained within a 3500 capillary array?
8 or 24 (24 at LSPCL)
What length array is used most in fragment size analysis on a 3500 instrument?
36cm
What is the minimum number of days that the anode and cathode buffer containers may be used for CE runs on a 3500?
14 days (manufacturer)
7 days (LSPCL)
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.
Detection
Excitation
Emission
What is the electrophoretic flow?
Electrophoretic flow is the movement of negatively charged DNA from the cathode to the anode.
What is the electroosmotic flow?
Electroosmotic flow is the movement of the anode buffer to the cathode.
What is electrophoresis?
The movement of charged particles through an electric field.
Describe the inner capillary environment.
-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.
How does the matrix affect the speed?
Larger particles move slower than smaller particles due to slowing movement through the matrix.
What are the 3 requirements for STR typing in CE?
Spectral resolution, spatial resolution, DNA sizing precision
During capillary electrophoresis, the data from the detector is measured in _______ and _______.
time and intensity
