Introduction
The purpose of the demonstration is to allow the students to get a "hands-on" opportunity to do a scientific experiment. The focus of the experiment should be getting as many students involved as possible, and to have fun with the experiment.
In the experiment, Blue Dextran is mixed with a dilute Phenol Red solution in a buffer containing sodium chloride and sodium acetate at pH 5.0. At this pH, the phenol red turns yellow (right in front of the students' eyes!). Upon mixing with Blue Dextran, the solution turns green. Application of the solution to a Pharmacia PD-10 column separates the green back into blue and yellow.
Materials and Methods
The following solutions will be needed:
* Blue Dextran, 3 mg/ml, in water
* Phenol Red, 10 mg/ml, in water
* 150 mM NaCl, 50 mM NaOAc, pH 5.0 (Equil. Buffer)
Note: you can purchase Blue Dextran and Phenol Red from Sigma Chemicals. Call 1-800-325-3010 for more information. Sigma product numbers are as follows: Blue Dextran/D5751 (5g/$66.80); Phenol Red/143-74-8(5g/$7.75)
The following equipment will be needed:
* Pharmacia PD-10 columns (at least 2)
Note: You can purchase PD-10 columns from Pharmacia (1-800-526-3593/ product number 17-0851-01/ Box of 30/$112.00)
* Powder funnels (1 per column)
* Ring stand with clamps
* P1000 and P20 Pipetmen/tips
* Disposable 12 x 75 mm test tubes
* Liquid waste container
* 25 ml or 50 ml plastic grad. cylinder
Experimental Procedure
1. Pre-equilibrate both PD-10 columns with at least 25 ml of equilibration buffer. This allows the second column to be used while with first column is being re-equilibrated.
2. Point out to the students beforehand that their eyes may play tricks on them. Let them think that magic is at work here. Add 1-2 ul of Phenol Red to 500 ul of equilibration buffer in a disposable 12 x 75 mm test tube. The solution turns yellow.
3. Ask the students if they know what color is made by the mixing of blue and yellow. Add 500 ul of Blue Dextran to the diluted Phenol Red solution to confirm their guess.
4. Transfer the green solution to the PD-10 column. Point out that the colors are separating from each other, but that only blue is visible at first.
5. Connect the powder funnel to the column. Add about 25 ml of equilibration buffer to the funnel and watch for the color separation. The students will notice complete separation of the blue from the yellow, with a white zone in between.
6. During chromatography, the students may have questions, and this is usually the best time to answer them, as well as to explain what is going on. A simple explanation of the theory is to compare the gel beads to "Wiffle Balls" and the difficulty or ease that big and small objects may have in fitting through the holes in the Wiffle Balls.
Labels: biochemistry, biotechnology, chromosomes, demonstration, genetic chromosomes, genetic engineering, project
