Course title

Pre-requisite

Course description

Approximately how much time per week do students spend conducting hands-on laboratory experiments in this course?
The Biotechnology class at Peoria High School meets every other day for ninety (90) minutes for an entire year; which equates to around 135 hours (90 days times 1.5 hours = 135 hours). Approximately 80 hours of that time is spent in the lab.

Briefly describe course content. Please include a list of the laboratory experiments or projects you do that require manipulation of equipment.
This course is designed to provide students with a hands-on laboratory experience in the field of Biotechnology. Students spend more than half of their time in the laboratory setting up; conducting and analyzing experiments. All solutions are formulated; pH adjusted; sterilized (when necessary); aliquoted and used by students. The labs performed in class are listed below:

Formulating Solutions
pH?ing Solutions
Formulating Tissue Culture Media
Plant tissue culture
Thin Layer Chromatography
Plating bacteria
Streaking Bacteria
Inhibition comparison
Epidemiology
Transferring colonies
Transfection of bacterial colonies
Electrophoresis
DNA extraction & isolation
Restriction Enzyme digestion and isolation of DNA
Protein extraction
Polymerase Chain Reaction amplification of DNA

Using standard Scientific Method outlined by the following questions; describe one typical laboratory assignment associated with this course.

State the problem or concept investigated during this laboratory assignment. (Do oranges stored in a refrigerator have more Vitamin C than oranges picked fresh from a tree?)
Am I homozygous (+); homozygous (-); or heterozygous (+/-) for the PV92 Alu gene insert on Chromosome 16?

Formulate a hypothesis for this problem using ?if/then? statements. (If oranges picked fresh from a tree have more Vitamin C; then juice from these oranges will take longer to turn a starch solution blue.)
If my DNA contains a band at 941bp then I possess the PV92 Alu insert.

Describe the experiment you performed to prove or disprove your hypothesis. List all essential materials. Describe each step you performed in the experiment.
1. Students used a saline solution and vigorously rinse their mouths to collect loose mucosal cells.
2. Cells suspensions were spun in a centrifuge and the supernatant was decanted.
3. The resuspended pellet was mixed with 20 µL of InstaGene Matrix and mixed well.
4. The suspension and Matrix were incubated at 56 ∫C for 10 minutes then shaken.
5. They were then incubated at 100 C for 5 minutes.
6. The suspension was again spun at 6000 x g for 5 minutes.
7. 20 µL of the supernatant was transferred to Polymerase Chain Reaction (PCR) tubes.
8. 20 µL of Master Mix (dNTPs; buffer; Taq DNA polymerase; PV92-Alu primers; and dye) was added and mixed.
9. The PCR tubes were placed in the Thermocycler and run through forty (40) cycles of:
1) 94 ∫C for 1 min
2) 60 ∫C for 1 min
3) 72 ∫C for 2 min
4) Repeat
10. A 10 minute 72 ∫C extension period was run following the cycles.
11. Following PCR the tubes were spun at 2000 x g for 3 seconds to concentrate the droplet.
12. 10 µL of loading dye (Xylene Cyanole and Glycerol) was added to each sample.
13. A 1% agarose gel using 1X TAE was loaded in the following manner:
1) 10 µL ?EZ Load? molecular mass ruler (1000bp; 700bp; 500bp; 200bp; 100bp)
2) 20 µL Student sample 1
3) 20 µL Student sample 2
4) 20 µL Student sample 3
5) 20 µL Student sample 4
6) 20 µL Student sample 5
7) 20 µL Student sample 6
8) 20 µL Student sample 7
14. Electrophoresis using the gel was run for 30 minutes at 100 Volts.
15. Following electrophoresis the gel was stained using BioRad ?Fast Blast?DNA stain at 100X concentration for 2 minutes under agitation.
16. The gel was destained using warm water through several rinses until DNA became visible.
17. The gel was photographed for analysis.
18. The lanes and bands were analyzed. Bands found that were around 941bp in size

Describe the results of your experiment or study. Use graphs and charts where appropriate.
The data presented represents seven students who conducted the experiment in the 2006-2007 Biotechnology class at Peoria High School.

Figure 1: Separation of DNA bands are shown for the gel in question. Bands were separated by size. Lane (1) contains the BioRad ?EZ Load? DNA molecular mass ruler (1000bp; 700bp; 500bp; 200bp; 100bp); (3); (4); & (7) homozygous positive (+/+); (6) & (8) homozygous negative (-/-); (2) & (5) heterozygous (+/-) with both the 641 and 941 bp fragments.

Explain your data or results. Give an analysis of your experiment.
As you can see in the Figure 1 above two of the individuals were homozygous positive (+/+) for the insert; three individuals were homozygous negative (-/-); and two individuals were heterozygous (+/-) showing bands at both 941 and 641 bp. The banding pattern was similar to that shown in the laboratory instructions.

The bands shown in the picture illustrate how the DNA migrated down the gel. The darker the stain each band shows indicates more DNA in that region. Although band darkness is not quantitative it does give a suggestion of amount. Some smearing can be seen above the bands; especially in the heterozygous samples. This is typical of most DNA electrophoresis and is seen in samples amplified through Polymerase Chain Reaction.

Note: In order to prevent student comparisons of disease or relatedness the samples were randomized prior to running. Students were assigned tubes of samples that may or may not have been their own and then asked to discuss their findings; hypothesis; and conclusions based on this new ?unknown? sample.

Write a conclusion for your study. Was your hypothesis supported or refuted?
The gel showed conclusively that members of the class were indeed different with respect to the PV92 Alu sequence. Although this particular gene sequence is not known to have correspondence to disease it is one of the bands used for identity by law enforcement officials.

Depending on the student?s hypothesis and the results of their sample they may discuss whether their statement was supported. Comparisons between one student?s sample and another can be made based on their ?random? samples.

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Date submitted

Approved

Approved competency code

• LBIO
• Biology

Approved date

Online / Virtual