DME Teaching Tips - Ideas for Further Explorations

HINT:  If  you are loading your home-made solutions or extracts into an agarose well, you have to increase their density so that they “sink” nicely into the well.  Otherwise, your solution will float away and not enter the well.  To increase the solution density, make your solution 5% sucrose or 5% glycerol.  For example, suppose that you have 100 mL of extract or solution – if you add 5 g. of sucrose to the 100 mL solution, this will make a 5% sucrose solution.  Alternatively, if using glycerol which is liquid, add 5 mL of pure (100%) glycerol into 95 mL of your extract or solution.

Biology

Using the setup of just the box (part I), use other chemical or natural pH indicators to explore; examples:

1. Beet Juice indicator:  (red in acidic, blue in basic).  Place four slices of fresh beet in pan with 1 cup water, heat till boiling and boil for about 5 minutes.  Cool.  Store red juice in dropper bottles.
2. Phenolphthalein:  find a laxative in drug store that contains phenolphthalein.  Mash four tablets, add about 10 ml of rubbing alcohol, let soak for 15 minutes, pour off liquid and store in dropper bottle.  (purple in very basic solutions, colorless in slightly basic and in acidic solutions)
3. Red cabbage juice (red in acid, green/blue in basic) – tear about five leaves of red cabbage into small pieces, place in one liter water, bring to boil and simmer till liquid turns deep purple.  Pour liquid through strainer or cheesecloth into storage bottle.  Keep refrigerated.
4. Phenol Red – make according to directions on label.

Using the setup of gel box and gel from part II, you can use food dyes or KoolAid from the grocery store or use extracts of naturally-colored materials to run on a gel – make predictions for charge and shape and test in the gel.  Don’t forget to make these extracts “dense” with sucrose or glycerol in the final solution as in the Hints section above.

Physics and Physical Science:

Use the gel and the dyes setup from part II, and

Measure the distance traveled over time (velocity = v) and derive the charge in coulombs.

Use ½ mv² = q v

+                                                                                     _

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   distance = d  (in meters – do conversion from mm)

Use a stopwatch to measure time of transit across d (in seconds). 

Use velocity  (v) = d/t

Kinetic Energy (K) = ½ m v²    where m is the mass in kg.

Conservation of Energy says that in the absence of external forces, all the K comes from a potential energy U.   Here U is the electric potential energy:  U = q V.  q is charge in coulombs and V is voltage in Volts (convert from mV).  Calculate K from calculating U.  Then q = K/V.

This value may be off since the gel is resistant to motion.  Hence, a further experiment could calculate changes in calculated q with different concentrations of agarose in the gel, i.e. different levels of resistance.

If you know the actual charge on the molecule, you can compare by using

% error =  q ^true – q ^measured/q ^true   x 100. 

A % error greater than 10 indicates that Energy Conservation is not valid.

Credit: Dr. Debra Burris, formerly of OCCC, now at University of Central Arkansas.

Chemistry:

For chemical reactions:

Make up the gel with TB buffer (leave out EDTA), cast the 2% agarose gel with two rows of wells as illustrated below.  For the wells closer to black negative pole, load one well on the left with calcium chloride and one well on the right with potassium phosphate.  For the wells closer to the red positive pole, load one well on the left with potassium phosphate and one well on right with calcium chloride.  If the ions migrate through gel to appropriate poles, predict what will happen when the ions meet:

                                    _                           

Write the reaction that you think is occurring:

What type of chemical reaction is this?

SETUP: 

Gel boxes with power supplies;  Use 0.8% agarose gels:  for one gel, mix 1 g. agarose with 125 mL of TBE buffer in 250 mL flask, stir over gentle heat to dissolve; pour when cool enough to hold in hand.

Bromothymol blue:  prepare concentrate by putting 0.4 g. of bromothymol blue powder into bottle with 100 mL volume – add 100 mL deionized or distilled water.  Mix well. To prepare sample bromothymol blue for electrophoresis, mix 2 mL of 0.4% BTB concentrate into 1000 mL of deionized or distilled water.  Store at Room Temperature.

Chemical Dye Stocks:  Pyronin Y, methyl orange, safranin O, Ponceau G, xylene cyanol, bromphenol blue, Janus Green.  Make 2% dye stock for each by measuring 0.2g of dye powder, add 10 mL deionized or distilled water, shake to dissolve, store at RT.  For sample dyes, do the following:

1. Prepare 50% glycerol soln by pouring 10 mL pure 100% glycerol into grad. Cylinder and add 10 mL deionized or distilled water – mix well – store in refrigerator.
2. To prepare sample dye for electrophoresis, obtain container that holds 25 ml.  Into container place 2 ml of 50% glycerol, 1 mL of the 2% dye stock, and bring to 20 mL volume with 17 mL of deionized or distilled water.  Repeat for each dye used. 
3. Aliquot for day of experiment into 12 ml aliquots.
4. Unknown dye:  2 mL of 50% glycerol, 1 mL each of 2% methyl orange, 2% bromophenol blue and 2% xylene cyanol.  BTV – add 15 mL deionized or distilled water – mix well. 

Exploratory materials:

5.      Beet Juice indicator:  (red in acidic, blue in basic).  Place four slices of fresh beet in pan with 1 cup water, heat till boiling and boil for about 5 minutes.  Cool.  Store red juice in dropper bottles.  Cannot store for long periods.

6.      Phenolphthalein:  find a laxative in drug store that contains phenolphthalein.  Mash four tablets, add about 10 ml of rubbing alcohol, let soak for 15 minutes, pour off liquid and store in dropper bottle.  (purple in very basic solutions, colorless in slightly basic and in acidic solutions).  Or make dye stock from powdered dye and then make into dye sample according to above protocol for other dyes.  Janus Green can also be made in this way.

7.      Red cabbage juice (red in acid, green/blue in basic) – tear about five leaves of red cabbage into small pieces, place in one liter water, bring to boil and simmer till liquid turns deep purple.  Pour liquid through strainer or cheesecloth into storage bottle.  Keep refrigerated; cannot store for long periods.

8.      Have some food dyes and Kool Aid samples ready for testing:  again, must add sucrose or glycerol to make a final conc. of 5% so the soln will sink into well- so for 10 mL of KoolAid, add 0.5 mL of pure glycerol or 250 ml of 50% glycerol or 0.5 g. of sucrose.

9.      TBE buffer:  for 1 Liter, use 1 g. of NaOH (mw 40.0), 108 g of Tris base (mw 121), 55 g. boric acid (mw 61.83), 7.4 g. EDTA (ethylene diamine tetraacetic acid, disodium salt, mw 372.24).

10.  Make TB buffer – use recipe for 1X TBE but leave off the EDTA in recipe.

11.  Make 2M saturated calcium chloride and 2M saturated potassium monohydrogen phosphate (separately) – 200 mL of each:  58.8 g calcium chloride  in 200 mL deionized or distilled water; 69.6 g. potassium phphphosphate, BTV 200 mL.  To prepare for loading into wells, take 100 ml of each, add 5 g. sucrose to each – label as “gel loading sample”.

Credits:  Some of the ideas for this laboratory exercise came from the University of Arizona Biotech project, CEPRAP at the University of California Davis , and Access Excellence.  Professor Debra Burris of OCCC contributed the physics exercise.

This material is based on upon work supported by the National Science Foundation under Grant #0202287 and NIH R25RR17282.  Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of NSF or NIH.