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DESKTOP ELECTROPHORESIS LAB - MOVING MOLECULES

INTRODUCTION:

Electrophoresis is a technique used for the separation of molecules by means of electric current. In this case, the molecules to be separated are placed in wells of an agarose gel medium which is submerged below the surface of a buffer. The molecules will move in the electrical field according to their charges. The positively charged molecules will move to the negative electrode (cathode) and the negatively charged molecules will move to the positive electrode (anode).

TARGET ABILITY LEVEL:

Regular, Honors or A.P. Biology

OBJECTIVE:

To build efficient electrophoresis systems for less than $15.00 each and use them to demonstrate electrophoretic ability of stain molecules and materials such as DNA.Th

TIME REQUIRED:

One day to build the boxes and power sources if each class takes up where the previous class left off. One class period to pour gels and one class period to load and run the gels (based on 55 minute class periods).

MATERIALS FOR BUILDING ELECTROPHORESIS SYSTEM:

Chamber

  • #10 or 11 Rubbermaid Container
  • 18 gauge (or so) seizing wire (purchased at local boat shop)
  • Microscope plastic slide box (lid or bottom)
  • Hot glue/hot glue gun
  • Dissecting Needle
  • Aquarium Sealant

Power Supply

  • 5 nine-volt batteries
  • 1 nine-volt battery clip (cut in half)
  • 2 alligator clips (1 black/1 red)
  • Scissors
  • Electrical or masking tape

SAFETY PRECAUTIONS:

Although 45 volts do not deliver enough current to be harmful to the human body, students should be instructed not to place fingers in the buffer solution and to close the containers while current is running. Use appropriate precautions in use of electrical gun, hot glue, and pointed objects.

Also, please refer to the appropriate MSDS sheets for the stains/dyes used.

PROCEDURE FOR BUILDING BOXES AND POWER SUPPLY:



  1. Secure one portion of the microscope slide box in the bottom of the Rubbermaid container with aquarium sealant, flat side up and open side down (using the lid requires less buffer). To produce a good seal, place sealant on all four edges of the slide box, place in appropriate position and press.
  2. Using a dissecting needle, make 2 holes through the wall of one side of the container just above the upper rim, and at least 1 cm to either side of the slide box platform. This orientation will be referred to as the front side of the box.
  3. Cut two pieces of wire about 4 cm longer than the width of the Rubbermaid container. Bend the end of the wire at a 90 degree angle where it touches the inside of the back side of the container. Mold the remainder of the wire so that the portion beyond the 90 degree bend runs toward the front of the box parallel to and approximately 0.5 cm to 1 cm above the floor of the container. Bend the wire at another 90 degree angle where it touches the inside of the front wall and another 90 degrees out to the outside of the box via the hole punched with the dissecting needle.
  4. Secure the wire at the original 90 degree bend to the back wall of the container with ample hot glue to prevent separation. Also seal around the wire where it emerges through the hole to the outside. ALLOW BOX TO DRY 24 HOURS, THEN CHECK FOR LEAKS.
  5. Bend the wires that stick out of the holes into loops for attachment of alligator clip leads from the power pack.



  6. To Make the Power Pack, arrange the 5 nine-volt batteries as shown in above figure. Carefully separate the wires of the nine-volt battery clip and snip with scissors into + and - leads. Attach the wires to the appropriate alligator clips (black to - and red to +) and secure by covering with plastic lever covers which are part of the original clips.

PART II: MOVING MOLECULES LAB (Addie Jackson) WL

MATERIALS:

  • 2 X 3 in. glass microscope slide
  • Index card and thick cardboard cut to fit glass slide (to build up slide to fit comb)
  • 3 X 5 index card
  • Pi-pump (Pipetor) to fit 10 ml pipet
  • 10 ml Pipet
  • Agarose gel (liquid, slightly warmer than room temperature)
  • Hot Plate
  • Electrophoresis chambers & power supplies built in Part I
  • Micropipet plungers and micropipets
    • Tris EDTA Borate Buffer, pH 8.2 or 8.6.
  • Stains: Any stains available to you may be used.

POURING THE GEL:

  1. Place a clean glass slide onto an index card and cut it to fit the glass slide.



  2. Fasten clips to both ends of the comb and align the comb in the middle of the slide so that the teeth of the comb just touch the glass. If teeth do not touch, build the slide up using as many pieces of cardboard as necessary. (see above figure - Equipment page)
  3. Carefully remove the white card underneath leaving a small space between the teeth of the comb and the surface of the glass slide. This insures a stable floor for each well.
  4. Using the pi-pump, aspirate 9 ml of warm (not hot) liquid agar into a pipet.
  5. Holding the pipet tip at a slight angle, release the agarose slowly but continuously on the surface of the glass slide, beginning around the teeth of the comb and working toward the edges of the slide, filling in as you proceed. The agarose will be held around the edges of the slide by surface tension if the agarose is carefully applied and is not forced over the edges. Try to avoid bubbles in the gel.
  6. Allow the gel to cool until it appears opaque; then pull up on one side of the comb, applying continuous but gentle pressure.
  7. The gel should remain on the slide throughout the electrophoresis. If the gel is not going to be used immediately, place it in a Zip-loc bag, blow in a small amount of air to cushion it, then zip it shut securely. The gels can then be stacked without damage and refrigerated until time for use.



  8. Place the gel on the platform in the electrophoresis chamber and pour the buffer over it until the gel is completely submerged. (See above figure - Electrophoresis Equipment diagrams)

LOADING THE GEL:



  1. Insert the metal plunger into the glass capillary tube micropipet See above figure - Electrophoresis Equipment diagrams) and aspirate 10 microliters of a dye sample into the pipet. Wipe the tip with a Kimwipe to remove excess.
  2. Carefully insert the tip of the micropipetor above and into the corner of a well. Depress the plunger slowly and carefully, filling the entire well. Continue for each well, using the plunger designated for each stain/dye.
  3. As you place each sample into its separate well, label a clean white index card, designating the stain in each well.

RUNNING THE GEL:

  1. Close the lid of the electrophoresis chamber.
  2. Attach leads (alligator clips) to wires on box and clip to power supply.
  3. Label the index card with positive (+) (red) and negative (-) (black) poles in relation to stains in the wells. Caution: Use homemade 45-volt power supplies with homemade boxes to prevent shock hazard. Do NOT use variable power supplies on which power can be cranked up to unsafe levels.
  4. Do not touch chamber or place fingers in buffer once power has been turned on.
  5. Allow the gel to run for 10 minutes. Disconnect the power. Remove the lid and observe the movement of the stains.

  6. Replace the lid and reattach the leads. Continue the electrophoresis until the fastest moving dye comes to within 1 cm of the end of the gel, or 30 minutes. Be sure to replace the correct lead to each wire as before: red to positive and black to negative.
  7. Turn off the power and remove the gel from the electrophoresis chamber. Using a spatula, place the gel on an index card. Label the stains.
  8. Measure the distance each stain migrated from the origin (well). Record the data in a table.

CONCLUSIONS:

  1. Which molecules were negatively charged?
  2. Which molecules were positively charged?
  3. What factors, other than charge, could have influenced the movement of these molecules?
  4. If an unknown was used, determine which stains were in the mixture, and give the supporting evidence from your data.

SOURCES FOR EQUIPMENT AND SUPPLIES

Modern Biology, Inc.
P.O. Box 97
Dayton, IN 47941-0097
1-800-733-6544

E-C Apparatus Corp
3831 Tyrone Blvd. North
St. Petersburg, FL 33709
1-800-EC RANGE

Helena Laboratories
1530 Lindbergh Drive
P.O. Box 752
Beaumont, TX 77704
1-800-231-2382

Sigma Chemical Corp.
P.O. Box 14508
St. Louis, MO 63178
1-800-325-3010



TEACHER INSTRUCTIONS


PREPARATION OF TRIS EDTA BUFFER SOLUTION: pH 8.2 - 8.6

Dissolve buffer crystals in distilled water as per instructions provided by vendor.

(Modern Biology buffer uses one 60 g package in water to produce 3000 ml of solution)

PREPARATION OF AGAROSE GEL:

  1. Dissolve 10 grams of agarose gel powder in 100 ml freshly prepared buffer solution.
  2. Heat the agarose-buffer mixture in a boiling water bath on a hot plate until the agarose is clear and boils gently for 2 minutes. Remove from hot plate and allow to cool only until container is comfortable to touch. At this point students should pour their gels.

WEIGHTING STAINS:

(If Necessary)

Stains may sometimes be less dense than the buffer solution. Hence, they will float to the surface rather than sink into the wells when applied. To check, place small amounts of buffer in small test tubes. Carefully apply a drop of the stain to the surface of the buffer solution. If the stain sinks, the stain is dense enough. If the stain disperses over the surface of the buffer, add a few drops of 1:1 glycerin:buffer solution. Reliable students may do this for you while you are calling roll!

SUGGESTED STAINS:

Methylene Blue, Methyl Green, Poncean Red, Safranin, Crystal Violet, Comassie Blue, Bromphenol Blue, Dye or Stain Mixture*, etc.

*Dye or Stain Mixture is suggested for comparison and identification of unknowns.

OTHER TIPS:

  1. Do not allow agarose gel to cool too long prior to use as it will begin to set up in the flask and will not pour smoothly.
  2. Distribute liquid agarose gel to students in 100 ml units contained in 250 ml flasks to facilitate pouring several gels at one time.
  3. Remind students to empty pipets while gel is still warm or it will set up in pipet.
  4. Prepare buffer a day in advance and keep under refrigeration. Gels run best with cold buffer.
  5. Pour buffer into gel chambers for students, to avoid excessive use on their part.
  6. For first time electrophoresis, Modern Biology sends everything prepackaged and provides excellent information/instructions for both teacher and student.
  7. BE SURE TO CHECK LOCAL LEGAL GUIDELINES OF YOUR SCHOOL DISTRICT FOR USE OF HOMEMADE ELECTRICAL APPARATUS IN THE CLASSROOM, IF ANY. Under NO circumstances should a commercial variable powersource be used on which power can be cranked up to unsafe levels.

INFORMATION REFERENCES

Addie Jackson
Westwood High School
12400 Mellow Meadow Dr.
Austin, TX 78750
(512) 250-1051


Woodrow Wilson Index


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