1. Biotech Lab #3 DNA Goes to the Races
“Gel electrophoresis”

2. 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.
Here is what a gel looks like….

3. The blue liquid is the DNA
Well being filled with DNA
Empty wells

4. What is happening? In gel electrophoresis, fragments that are
smaller move faster and further in the gel
than the larger fragments. Can you id the
Direction in which the fragments are moving?
Gel electrophoresis separates according to size which is base pairs.

5. Let’s watch how a gel electrophoresis is done.
Gel Electrophoresis Animation
Dolan Center Animation with no audio

6. DNA Molecular Structure
Why is this slide in this power point?
Click to read the next slide.

7. Because gel electrophoresis box has a positive and negative end, DNA will move through the gel. Why? It has to do with the fact that opposite charges attract. Read the info box on DNA.
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.
The negative part of DNA is
attracted to the positive end of the
gel electrophoresis box.

8. The following slides follow the process in order….
Performing Gel Electrophoresis
The following slides follow the process in order….

9. 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.

10. 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.

11. 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.

12. Where does the DNA move?
As the separation process continues, the separation between the larger and smaller fragments increases.

13. 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

14. DNA goes to the Races Lab Objectives:
Review the different types of restriction enzymes and notice how they can produce different size fragments.

15. Actual DNA Electrophoresis Equipment
The first step is to prepare a tray to hold the gel matrix (agarose).
The ends of the tray are taped.

16. 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.

17. 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.

18. 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.

19. 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.

20. 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.

21. Can you explain these pictures?

22. Gel Pic Analysis

23. Gel Picture

24. Gel Pic Gone Wrong

25. Intercalation