18 Questions
What determines the amount of time needed for electrophoresis?
Length of the gel, voltage used, and sizes of the molecules in the sample
What is the purpose of DNA ladders containing tracking dyes in gel electrophoresis?
To monitor gel runs and ensure bands of interest are not masked by dyes
When should electrophoresis be stopped in relation to the migration of bromophenol blue dye?
Approximately when it has migrated 60% of the gel length
Why is monitoring run time crucial in electrophoresis?
To prevent smallest molecules from migrating off the gel
What happens if run times are shorter than necessary in electrophoresis?
Bands will not be completely resolved
How do DNA ladders with tracking dyes contribute to gel electrophoresis?
Help monitor gel runs and ensure bands of interest are visible
What is the typical range for the voltage set in nucleic acid electrophoresis?
5-10 V/cm
What is the effect of low voltage in DNA electrophoresis?
Poor band resolution and diffusion
Why is it important to monitor run time during electrophoresis?
To ensure proper band resolution
What does it mean if the smallest molecules in the sample migrate off the gel during electrophoresis?
Insufficient band resolution
What problem can occur if high voltage is applied during DNA electrophoresis?
"Smiling"
How does constant voltage assist in running an electrophoresis gel?
Aids in proper band resolution
What is the main purpose of gel electrophoresis in molecular biology?
To identify, quantify, and purify nucleic acids
Which type of support matrix is ideal for separating macromolecules like nucleic acids and protein complexes?
Agarose
What property of a biological molecule influences its mobility through an electric field during electrophoresis?
Shape of the molecule
In polyacrylamide gel electrophoresis, what is the main advantage of using a smaller pore size?
Enhanced resolution of smaller molecules
Which factor does NOT affect the mobility of a biological molecule through an electric field during electrophoresis?
Temperature of the solvent
Why is agarose gel preferred for separating macromolecules like nucleic acids and protein complexes rather than polyacrylamide?
Agarose has a higher resolving power
Study Notes
Running Electrophoresis
- Constant voltage is commonly employed in nucleic acid electrophoresis, with voltage typically set at 5-10 V/cm.
- The voltage to be applied (V) is equal to the distance between the electrodes (cm) multiplied by the recommended V/cm.
- Low voltage results in poor band resolution and diffusion, while high voltage can cause "smiling".
- Run conditions based on fragment size determine the amount of time needed for electrophoresis.
Run Time
- The length of the gel, voltage used, and sizes of the molecules in the sample determine the run time.
- Electrophoresis is usually run until the band of interest has migrated 40-60% of the gel length.
- Run time should be monitored to ensure the smallest molecules in the samples or standards do not migrate off the gel.
- DNA ladders containing tracking dyes can help monitor gel runs and ensure bands of interest are not masked by the dyes.
Electrophoresis
- Electrophoresis is the transport of charged molecules through a solvent by an electric field.
- Any charged ion or molecule will migrate when placed in an electric field.
- Mobility of a biological molecule depends on field strength, net charge, size and shape of the molecule, ionic strength, and properties of the matrix.
Support Matrix
- Two common support matrices used in electrophoresis are polyacrylamide and agarose.
- The support matrices act as porous media and behave like a molecular sieve.
- Separation of molecules depends on the gel pore size of the support matrix used.
- Agarose has a large pore size and is ideal for separating macromolecules, while polyacrylamide has a smaller pore size and is ideal for separating smaller molecules.
Learn the fundamental concepts of electrophoresis, which involves the movement of charged molecules through a solvent using an electric field. Understand how this technique is used for analyzing and separating proteins and nucleic acids based on their charge density.
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