Agarose Gel Flashcards
What is DNA gel electrophoresis?
Running DNA on an agarose gel using an electrical current to separate the bands or fragments.
What is a band or fragment?
Bands/fragments are composed of many pieces of DNA of the same length.
The pieces in a particular band are usually identical to one another, meaning they all have the same sequence of nucleotides.
What is DNA control?
DNA control is always electrophoresed with the other DNA.
It contains bands in which the size is actually known. This allows you to determine the sizes of the band in the DNA of interest.
You take a plasmid sample that contained 4 million copies of the plasmid. The plasmid is digested and there are two bands observed. How many pieces of DNA will be in each band?
There will be 4 million pieces of DNA in both strands
How many pieces of DNA will be in two bands if two plasmids were digested?
There will be two pieces of DNA in both strands`1
What does the band size represent in gel electrophoresis?
The size of the band is proportional to how thick the band or fragment is in the same lane in relation to the other bands.
Why is DNA gel electrophoresis done?
Gel electrophoresis allows you to separate bands and determine their sizes.
This is done when you have isolated a plasmid from a bacterium and you want to make sure the plasmid has not been altered. The plasmid should have the restriction enzyme profile on the gel that matches up to the restriction enzyme map.
What is the rationale behind gel electrophoresis?
It involves the chemistry of the DNA backbone and how it causes the DNA to migrate to the positive electrode during gel electrophoresis.
What is the gel in gel electrophoresis?
The gel used is agarose gel, which has wells where the DNA samples are placed.
It is like a thick jello that contains a buffer that allows it to conduct electricity. The gel is placed in a buffer that also conducts electricity.
DNA will run towards the positive electrode based on size. The bands on the gel can be visualized and their sizes can be determined.
Where will the smallest bands be? Should they be more intense than the larger ones?
The smaller bands should be towards the bottom. They should be less intense than the larger bands because, although they contain the same amount of DNA, the size is smaller.
What are the steps in performing gel electrophoresis?
- Pouring the gel
- Load the DNA sample in the wells of the gel with loading dye
- Run the gel using an electric current
- Visualize the gel under UV light and photograph it
Step 1: Pouring the gel
Involves pouring melted agarose into a gel tray and a comb is used to make the wells.
Peacock’s solution will be used as a buffer when preparing the melted agarose.
What is Peacock’s solution?
It is a buffer that contains a salt that provides ions that allow electricity to be conducted through the gel.
Step 2: Loading the DNA gel
The same buffer in the gel is poured into the gel box. The gel and tray are placed in the gel box. (so like.,., gel box contains gel and tray which is submerged in buffer).
The DNA samples are mixed with loading dye and pipetted into the wells on the gel.
What are the purposes of the loading dye?
- The loading dye weighs down on the DNA so it remains in the well and does not float out before the gel is hooked up to current
- It creates a dye front when the gel is running so you can figure out how far the DNA has run on the gel. Some loading dyes contain more than one dye.
