Biotech Lab #5 DNA Goes to the Races

“Gel electrophoresis”

What is this biotech principle?

Gel electrophoresis is a procedure for separating a mixture of molecules through a stationary material (gel) in an electrical field.

DNA Molecular Structure

Organic molecules such as DNA are charged. DNA is negatively charged because the phosphates (red circles) that form the sugar-phosphate backbone of a DNA molecule have a negative charge.

What is happening?

In gel electrophoresis, fragments that are smaller move faster and further in the gelthan the larger fragments

Gel electrophoresis separates according to size which is base pairs.

Gel Electrophoresis Animation

Dolan Center Animation with no audio

DNA Fingerprinting

Let’s watch how a gel electrophoresis is done.

Where will we put our extracted and digested DNA sample?

• Gel is prepared which will act as a support for separation of the fragments of DNA. The gel is a jello-like material, usually agarose, a substance derived from seaweed.

• Holes are created in the gel. These will serve as a reservoir to hold the DNA solution.

 

How do the fragments migrate?

• The gel matrix acts as a sieve for DNA molecules. Large molecules have difficulty getting through the holes in the matrix. Small molecules move easily through the holes

• Because of this, large fragments will lag behind small fragments as DNAs migrate through the gel.

What is will the DNA sample be like?

DNA solutions (mixtures of different sizes of DNA fragments) are loaded in a well in the gel.

Where does the DNA move?

As the separation process continues, the separation between the larger and smaller fragments increases.

How can we determine the fragment size of the DNA?

• Molecular weight markers are often electrophoresed with DNAs.

• Molecular weight markers are usually a mixture of DNAs with known molecular weights

• Molecular weight markers are used to estimate the sizes of DNA fragments in your DNA sample

Paper Model of a Gel

At the end of this lab, you will have a paper model of a gel that contains three digested DNA samples. You must properly place the different fragments for each enzyme in the proper location on the gel. Once you have had your gel checked, your teacher will give you another task: add the marker.

Actual DNA ElectrophoresisEquipment

The first step is to prepare a tray to hold the gel matrix (agarose).

The ends of the tray are taped.

 

What are the other parts are needed?

A "gel comb" is used to create holes in the gel.

The comb is placed in the tray.

What goes into the gel box?

• Agarose powder is mixed with a buffer solution, usually tris borate EDTA (TBE buffer). The solution is heated until the agarose is dissolved.

• The hot agarose solution is poured into the tray and allowed to cool.

How is the DNA placed in the gel box?

• After the gel is cooled, tape is removed from the ends of the gel tray and the gel tray is placed in an electrophoresis chamber.

• The electrophoresis chamber is filled with buffer, covering the gel. This allows electrical current from poles at either end of the gel to flow through the gel.

• Finally, DNA samples are mixed with a "loading dye". The loading dye allows you to see the DNA as you load it and contains glycerol or sucrose to make the DNA sample heavy so that it will sink to the bottom of the well.

What do we do with the DNA sample?

• A safety cover is placed over the gel (to keep you from frying yourself) and electrodes are attached to a power supply. Electrical current is applied.

• DNA fragments will migrate through the gel at various rates, depending on their size.

• When the dye marker indicates that DNA fragments have moved through the gel, the current is turned off and the gel is removed from the tray.

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Actual Gel Picture

• DNAs are visualized by staining the gel with ethidium bromide which binds to DNA and will fluoresce in UV light.

• This photograph is of various types of DNA that have been electrophoresed on the same gel. Note that high molecular weight DNAs do not separate well on this gel. This can be corrected by altering gel density.

Can you explain these pictures?

Gel Pic Analysis

Gel Picture

Gel Pic Gone Wrong

Intercalation