Gel Electrophoresis Flashcards
What is electrophoresis?
This is the movement of molecules by an electric current
What charge does DNA have and why?
DNA is negatively charged. This is due to its phosphate groupsb
Towards what pole does DNA move to?
The positive pole (anode)
How is DNA electrophoresis carried out?
DNA is applied to a gel matrix, and then e,extricate current pulls the DNA through that gel matrix which slows its progress towards the positive side
What is therefore the relationship between DNA speed and its size ?
DNA trace,s at speeds inversely related to its size. Big DNA molecules go slower while smaller ones GI faster and migrate further in the gel
Why are gels used for electrophoresis?
Gels form a porous matrix that the DNA can squeeze through
What are the two most common gels used in molecular biology?
Agarose
Polyacrylamide
What us agarose gel?
It is a polymer from seaweed and a component of the agar used for bacterial culture
Agarose is used for what type of DNA molecules?
Larger DNA molecules of 50bp to 50000bp
What does the concentration of agarose determine?
The sizes of the spaces in the gel
What kind of pores do high concentrations of agarose make?
Smaller spores
Low concentration of agarose results in what type of pores?
High pores
In what concentrations is agarose used?
0.5-5%
High percentages of agarose are used for
Smaller DNA molecules
Low percentages of agarose are used to resolve
Larger DNA molecules
Polyacrylamide gel electrophoresis is used to resolve what type of DNA fragments?
Very small DNA fragments and ss DNA down to 1bp
PAGE is often used for what type of analysis?
Sequencing and mutation analysis
In what direction is PAGE typically run? Why?
Vertically, it’s a potent neurotoxin
What’s the first step when making an agarose gel?
Making the electrophoresis buffer
What is the use of buffer in electrophoresis?
The buffer carries the current and protects the samples during electrophoresis
What are the two most common types of buffer? And how how do they differ?
Tris acetate and Tris borate. Tris acetate moves faster but with a smaller buffering capacity while Tris borate moves slower with a better buffering capacity
What does the loading dye added to the DNA sample do?
Gives color to DNA for easier visualization
Makes DNA denser than water so it stinks to bottom of well
Has tracking dyes that separate during electrophoresis to indicate progress of electrophoresis
What is a molecular weight standard?
It is a concentrated control stock of DNA fragments of known size
