Agarose Gel Electrophoresis Flashcards
What is agarose gel?
It is made out of seaweed that solidifies at room temperature into a gel slab.
How is agarose gel used in Agarose Gel Electrophoresis?
The gel slab acts like a molecular sieve as it has many minute pores which the DNA fragments can squeeze through.
Why is the relationship of smaller DNA fragments?
The smaller the DNA fragments, the easier and faster they migrate through the gel and hence are furthest away from the well.
What is Agarose Gel Electrophoresis?
A common laboratory method for separating DNA fragments of various sizes.
Why do DNA fragments move?
DNA fragments are negatively charged. The electrode placed near to the wells containing the DNA sample is negatively charged
The DNA fragments will be repelled but attracted to the positive terminal at the far end of the gel.
How do the DNA fragments move?
The agarose gel has tiny pores which the DNA fragments have to squeeze through as they migrate towards the other end of the gel.
In a sample, the DNA fragments are of different lengths, hence different size and weight.
The longer (heavier) DNA fragments will move slower than the shorter ones as they experience greater obstruction
DNA fragments of the same length will move at the same speed and hence forming a band at the end of the experiment.
Each band contains many DNA fragments of the same size and weight.
How does one make the gel slab?***
Agarose gels are made by first boiling a mixture of powdered agarose and a buffer stabilizes the pH
When the mixture is cooled to about 60C the solution is poured into a gel tray, inserted with a comb.
When further cooled to room temperature, the agarose solidifies to produce the gel with indentations called wells (created by the comb)
The gel together with the tray is then loaded into the electrophoresis chamber.
How to set up the gel chamber?
A buffer solution is added into the chamber till the tray with the gel is just submerged in it.
The negative terminal is attached to the end near the wells, and the positive terminal at the far end.
What do loading dyes contain?
Glycerol
Sucrose
What are the three functions of loading dye?
- The loading dye makes the DNA sample visible for easy loading.
- The leading dye helps track the migration of the DNA fragments (since DNA is colourless) during electrophoresis
- The glycerol/sucrose which is a dense liquid weighs/sinks the DNA sample into the well during loading so that it will not float away and becomes lost.
Explain in detail the process of the Migration of DNA fragments
After the wells are filled, the power supply is turned on.
An electric charge is transferred through the gel
The negatively charged DNA fragments are repelled by the negative electrode but attracted to the positive electrode.
At this stage, the DNA bands cannot be seen but the tracking dye (dark blue) allows the progress of electrophoresis to be followed.
How does the tracking dye allow us to track the migration of the DNA bands?
The smallest DNA fragments typically follow behind the blue dye down the gel towards the +ve terminal.
How to measure the size of DNA fragments?
This is done using the DNA ladder, also known as Molecular Mass Ruler.
what is a DNA Ladder?
It is a mixture of DNA fragments of known sizes.
How is DNA ladder used to measure samples?
The DNA ladder is loaded in a separate well and run alongside with the DNA samples.
It forms the ruler that can be used to estimate size of the unknown DNA fragments in other Gel lanes
