Name:                                                                         Block:                         

IB Gel Electrophoresis Introduction Lab

Gel electrophoresis is a basic biotechnology technique that separates molecules according to their size and charge.  It is frequently used to analyze and manipulate samples of DNA, RNA, or proteins.  In this laboratory activity, agarose gel electrophoresis will be used to separate and characterize colored dye molecules of various sizes and charges.  

In gel electrophoresis, samples to be separated are applied to a porous gel medium made of a material such as agarose.  Agarose is a purified form of agar, a gelatinous substance extracted from red algae.  Agarose gels are made by first adding powdered agarose to liquid buffer and boiling the mixture until the agarose dissolves.  This molten agarose is then cooled to about 55-60 degrees Celsius, poured into a gel mold called a casting tray, and allowed to solidify.  Before solidification occurs, a comb is placed in the casting tray to create a row of wells into which samples are loaded once the comb is removed from the solidified gel.

                                                                           

Once all the samples have been loaded into the wells, the chamber is connected to a power supply and an electrical current (usually 50–150 V) is applied to the gel. The chamber is designed with a positive electrode (anode) at one end and a negative electrode (cathode) at the other end. Electrophoresis literally means “to carry with electricity;” once the electric field is established, charged molecules in the samples migrate through the pores of the gel toward their pole of attraction. Molecules with a net negative charge migrate toward the positive electrode and molecules with a net positive charge migrate toward the negative electrode. The overall charge of a molecule affects the speed at which it travels through the gel. Highly charged molecules migrate more quickly through the gel than weakly charged molecules.

                        

The mobility of a molecule during gel electrophoresis also depends on its molecular size and shape. The small pores of the gel matrix act as a sieve that provides great resolving power. Small molecules maneuver more easily through the pores than larger molecules and therefore travel relatively quickly. Large molecules encounter more resistance as they make their way through the tiny pores and therefore travel at a slower rate.

                        

Size and net charge are factors that together determine how quickly molecules will travel through the gel, and thus what their migration distance will be. Small size and strong charge increase a molecule’s migration rate through the gel. Large size and weak charge decrease the migration rate. (Note: In electrophoresis of DNA, since all the samples have the same charge, their migration rate is based solely on size).

                        

In this activity, five known dye samples and three unknown dye mixtures will be subjected to agarose gel electrophoresis. Some of the dyes will be attracted to the negative electrode and some to the positive electrode, depending on their overall charge. Each of the known dyes will exhibit a unique gel migration distance that relates to its molecular size and net charge. Students will identify the components of the unknown dye mixtures by comparing the migration distances and direction of migration of the unknown dyes to those of the known dye samples[1].

        

        

Materials:

Procedure:

Part A: Casting the Agarose Gel

  1. Heat the 0.8% agarose solution so that it is a liquid; careful, it’s hot!  Let it cool a bit but not solidify.
  2. Put a rubber stopper on either end of a 7x7cm gel tray.
  3. Add the 0.8% agarose solution to the casting tray; the liquid should be just below the top of the rubber stoppers. 
  4.  Insert the well-forming comb in the middle set of grooves in the casting tray in the middle position. This will create a row of wells in the middle of the gel once the gel has formed.
  5. Allow the gel to sit undisturbed while it solidifies. Be careful not to move or jar the casting tray during this time. Once the agarose has hardened, the gel will appear cloudy. The gel will solidify in 10–15 minutes.
  6. Once the agarose has solidified, slowly and carefully remove the comb from the gel without tearing the wells; remove the rubber stoppers as well.  Place the casting tray and the gel into the electrophoresis chamber oriented with the positive end of the tray towards the positive (red) end of the electrophoresis chamber.
  7. Fill the electrophoresis chamber with 1× TBE (tris-borate-EDTA) buffer to a level that covers the surface of the gel.
  8. Make sure the sample wells left by the comb are completely submerged in 1× TBE buffer. If “dimples” appear around the wells, slowly add more 1× TBE buffer until they disappear.
  9. The gel is now ready to load with dye samples. If you will be loading the gel at another time, cover the electrophoresis tank with the lid to prevent the gel from drying out

Part B: Loading the Gel

Dye samples will be loaded using 5-50 μL micro pipets.  Micro pipette provide very accurate dispersal of micro volumes and are commonly used in real laboratories.

  1. Set the pipet loading volume to 20 μL; this will be the volume of dye loaded into each well of the gel.
  2. Put a yellow micro pipet tip on the micropipet; be sure to close the pipet tip box when done so as to avoid contamination.
  3. Press the plunger down until it stops, place tip in dye sample, and release plunger.  You have now collected 20 μL in the micropipet.
  4. Using your dominant hand, steady the pipet over the well above the buffer layer of the first well on the left hand side. Rest the elbow of your dominant arm on the lab bench to stabilize your hand.  Expel any air from the pipet so that they dys is at the very tip.  Using your non-dominant hand, guide the pipet through the surface of the buffer and position it directly over the well.  See Figure 1 below for visual explanation.
  5. Very carefully and slowly expel the dye by pushing the plunger all the way down while it hovers just above the well; the dye will sink to the bottom of the well.  Before releasing your hand from the plunger, remove the pipet tip from the liquid buffer.        
  6. Repeat this process for each of the dye samples, continuing from left to right in the order shown below.  Use a clean plastic pipet tip for each dye sample        

Order of Dyes in Wells:

  1. Bromphenol Blue
  2. Methyl Orange
  3. Ponceau G (red)
  4. Xylene Cyanol
  5. Pyronin Y
  6. Unknown

Part C: Gel Electrophoresis                                                

  1. Once all the dye samples have been loaded, place the lid on the electrophoresis chamber. Orient the lid with the positive end of the chamber connected to the red (positive) cord and the negative end of the chamber connected to the black (negative) cord. Then connect the electrical cords to the power supply, with the positive lead in the positive input (red to red) and the negative lead in the negative input (black to black). If using a multi-channeled power supply, make sure both electrical leads are connected to the same channel.        
  2. Turn on the power supply and set it to the desired voltage of 125. Watch as the dyes slowly move into the gel and separate over time. Do not allow any of the dyes to run off the gel.  Allow the gel to run for approximately 15-20 minutes.
  3. Once the desired separation of dyes has been achieved, turn off the power, disconnect the leads from the inputs, and remove the top of the electrophoresis chamber.        
  4. Carefully remove the casting tray and slide the gel into the plastic tray for observation and analysis.

Part D: Analysis & Conclusion

  1. In the table below, record the number of dye bands in each lane and the direction of migration (positive or negative) for each band.  Determine the migration distance of each dye in the known and unknown samples by measuring the distance from the center of the starting well to the center of the dye band with a ruler.  

Lane

Sample

Number of Bands

Direction of Migration

Migration Distance (cm)

1

2

3

4

5

6

  1. Which dye molecule traveled farthest through the gel and which traveled the shortest distance?  What properties affect migration distances?

  1. What was the charge of the dye molecules that migrated toward the positive electrode and of the dye molecules that migrated toward the negative electrode?  How do you know; describe using data?

  1. Why is electrical current necessary for separating molecules by electrophoresis?

                                                          


[1] Laboratory procedure and background adapted from Wards Introduction Gel Electrophoresis Amazing Dyes Manual