11 Questions
What role does ethidium bromide play in gel electrophoresis?
It intercalates with DNA, allowing fragments to be visualized under UV light.
In gel electrophoresis, which direction do negatively charged DNA fragments move?
Towards the anode, due to its positive polarity.
Why do smaller DNA fragments travel faster through the gel during electrophoresis?
They have lower molecular weight and can navigate through the pores more easily.
What effect does ultraviolet light have on DNA fragments stained with ethidium bromide?
It causes the DNA fragments to fluoresce.
How are particles separated in gel electrophoresis?
By their charge, with negatively charged particles moving to the anode and positively charged particles moving to the cathode.
Which of the following is NOT an application of agarose gel electrophoresis?
Determination of protein structure
What is used to confirm the current flow during electrophoresis?
Bubbles coming off the electrodes
Towards which electrode does DNA migrate during agarose gel electrophoresis?
Positive electrode (red)
What component is mixed with DNA samples before loading them into the wells?
Loading buffer
Which method can be used to judge the migration of DNA in the gel?
Monitoring tracking dyes
Before Northern analysis, which type of biomolecule is separated using agarose gel electrophoresis?
RNA
Study Notes
Gel Electrophoresis
- In gel electrophoresis, negatively charged particles move towards the anode, while positively charged particles move towards the cathode.
Separation of DNA Fragments
- Gel electrophoresis separates DNA fragments by size in a solid support medium, such as agarose or polyacrylamide.
- The process involves loading a DNA sample into sample wells, followed by the application of an electric current.
- The negatively charged DNA then migrates towards the anode (positive) end.
- The rate of migration is proportional to size, with smaller fragments moving more quickly and winding up at the bottom of the gel.
Staining and Visualization of DNA
- DNA is stained by including the fluorescent dye, ethidium bromide, in the gel or intercalating it.
- As DNA fragments migrate through the gel, they take up the dye.
- Ultraviolet light causes the intercalated dye to fluoresce.
- The larger fragments fluoresce more intensely, although each fragment of a single class of molecule is present in equimolar proportions.
Agarose Gel Electrophoresis
- Agarose gel electrophoresis is a routine method for separating proteins, DNA, or RNA
- Applications of agarose gel electrophoresis include:
- Estimating the size of DNA molecules
- Analyzing PCR products
- Separating restricted genomic DNA prior to Southern analysis
- Separating RNA prior to Northern analysis
- Estimating the size of DNA fragments after digesting with restriction enzymes
Preparing and Performing Electrophoresis
- To prepare the gel, samples containing DNA are mixed with loading buffer and pipetted into the sample wells
- The lid and power leads are placed on the apparatus
- A current is applied, and a current flow can be confirmed by observing bubbles coming off the electrodes
- During electrophoresis, DNA will migrate towards the positive electrode, usually colored red
- The distance DNA has migrated in the gel can be judged by visually monitoring migration of tracking dyes
Learn how gel electrophoresis separates DNA fragments by size in a solid support medium, using an electric current to migrate negatively charged DNA towards the anode.
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