Gel Electrophoresis (Shayleen Scantlin)



Author

Shayleen Scantlin (Biology and AP Biology Teacher at Panorama High School)

Principles

The gel electrophoresis equipment can show many different principles. It can be used for molecular biology to separate out strands of DNA and RNA. This can be used to solve crimes, determine paternity and much more. In chemistry it can be used to sort out proteins and nanoparticles. 



Standards

Biology Grades 9-12

5. The genetic composition of cells can be altered by incorporation of exogenous DNA into the cells. As a basis for understanding this concept:

a.            Students know the general structures and functions of DNA, RNA, and protein.

d.* Students know how basic DNA technology (restriction digestion by endonu­ cleases, gel electrophoresis, ligation, and transformation) is used to construct recombinant DNA molecules.

Investigation and Experimentation Standards:

1. Scientific progress is made by asking meaningful questions and conducting careful investigations. As a basis for understanding this concept and addressing the content in the other four strands, students should develop their own questions and perform investigations. Students will:

a. Select and use appropriate tools and technology (such as computer-linked probes, spreadsheets, and graphing calculators) to perform tests, collect data, analyze relationships, and display data.

l. Analyze situations and solve problems that require combining and applying concepts from more than one area of science.

Materials needed

  • 250 mL of TBE Buffer
  • 2.85 L of Distilled water
  • 1% agarose solution
  • Water bath or microwave
  • Flask
  • CarolinaBLU stain
  • comb
  • gel box
  • DNA samples
  • masking tape
  • gel electrophoresis chamber
  • power supply
  • needle point pipets
  • light box

Procedure

Preparing the agarose gel

  Measure 1.25 g Agarose powder and add it to a 500 ml flask

  Add 125 ml TAE Buffer to the flask.  (the total gel volume well vary depending on the size of the casting tray)

  Melt the agarose in a microwave or hot water bath until the solution becomes clear.  (if using a microwave, heat the solution for several short intervals - do not let the solution boil for long periods as it may boil out of the flask).

  Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly. 

  Seal the ends of the casting tray with two layers of tape.

  Place the combs in the gel casting tray.

  Pour the melted agarose solution into the casting tray and let cool until it is solid (it should appear milky white).

  Carefully pull out the combs and remove the tape. 

  Place the gel in the electrophoresis chamber.

  Add enough TAE Buffer so that there is about 2-3 mm of buffer over the gel.


Loading the gel

  Add 6 ml of 6X Sample Loading Buffer to each 25 ml PCR reaction

  Record the order each sample will be loaded on the gel, including who prepared the sample, the DNA template - what organism the DNA came from, controls and ladder.

  Carefully pipette 20 ml of each sample/Sample Loading Buffer mixture into separate wells in the gel.

  Pipette 10 ml of the DNA ladder standard into at least one well of each row on the gel.

Running the gel

  Place the lid on the gel box, connecting the electrodes. 

  Connect the electrode wires to the power supply, making sure the positive (red) and negative (black) are correctly connected.  (Remember – “Run to Red”)

  Turn on the power supply to about 100 volts.  Maximum allowed voltage will vary depending on the size of the electrophoresis chamber – it should not exceed 5 volts/ cm between electrodes! 

  Let the power run until the blue dye approaches the end of the gel.

  Turn off the power.

  Disconnect the wires from the power supply.

  Remove the lid of the electrophoresis chamber.

  Using gloves, carefully remove the tray and gel.

Gel Staining

  Using gloves, remove the gel from the casting tray and place into the staining dish.

  Add warmed (50-55°) staining mix.

  Allow gel to stain for at least 25-30 minutes (the entire gel will become dark blue).

  Pour off the stain (the stain can be saved for future use).

  Rinse the gel and staining tray with water to remove residual stain.

  Fill the tray with warm tap water (50-55°).  Change the water several times as it turns blue.  Gradually the gel will become lighter, leaving only dark blue DNA bands.  Destain completely overnight for best results.

  View the gel against a white light box or bright surface.

  Record the data while the gel is fresh, very light bands may be difficult to see with time.

Explanation

During electrophoresis, the gel is submersed in a chamber containing a buffer solution and a po

sitive and negative electrode.  The DNA to be analyzed is forced through the pores o


f the gel by the electrical current.  Under an electrical field, DNA is negative and will move to the positive electrode (red) and away from the negative electrode (black). The DNA will be visualized by the use of a dye that binds to DNA. The smaller pieces will travel farther and the longer pieces will move slower. 

People have different lengths of their DNA because everyone has different junk DNA. This will revel different patterns. A change in the DNA sequence will change where the restriction cut site will be and different size pieces of DNA will be revealed. 




Questions

1. How would it affect the spread of the bands to use a gel of increased concentration?

The tighter gel matrix reduces effective separation of larger fragments but more effectively will separate the smaller DNA fragments. 

2. If the two suspects had been identical twins, how would it have influenced the results?

The two suspects would be so alike that it might not be possible to determine which one committed the crime. 

3. If more than two rows of bands look alike, what could have happened during the gel-loading?

Someone may have forgotten to change tips/ pipets between samples which will mix the DNA in one well. 

4. Why do some DNA fragments move farther than others?

The shorter fragments will move farther. 

5. Why do people have differences in their band pattern?

This is due to junk DNA and having differences at where the restriction sites are cut.

Everyday examples of the principles illustrated

People use this technology everyday when looking at forensic evidence for crimes. This technology can put people behind bars and free people that were wrongfully acused. People also use this for paternity testing. This also can be used for medical diagonosing. Plus it can show evolutionary relationships between species. 

Photos

Movies

YouTube Video

References

http://www.phschool.com/science/biology_place/labbench/lab6/concepts2.html
http://sciencefair.math.iit.edu/techniques/gelelectrophoresis/
http://www.youtube.com/watch?v=BDadjDRl-Vg
http://www.youtube.com/watch?v=6_4AY3lYRgo&feature=related
http://www.carolina.com/text/teacherresources/instructions/biotech/pcr_forensics_student_guide.pdf



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