DNA Fingerprinting Supplies from Iowa State Student Preparation DNA and Enzyme Preparation

1. DNA Fingerprinting Supplies from Iowa State Student Preparation DNA and Enzyme Preparation

2. Obtaining Supplies (Iowa Teachers Only) Teachers should request the following supplies (next slide) at least a week in advance. Supplies should be ordered by contacting Lori Miller by phone in Iowa, 1/800-643-9504 or 515/294-9818, or by Fax: 515/294-4629. The electrophoresis equipment can be obtained by contacting the Area Education Agency. DNA Fingerprinting

3. Supply List 6 boxes of pipette tips (already autoclaved) 34 µl of 0.025 µg/µl pBR322 per class section (minimum of 68 ml per school) 17 µl of 0.075 µg/µl Ad-2 DNA per class section (minimum of 34 ml per school) 17 µl of 0.15 µg/µl l DNA per class section (minimum of 34 ml per school) 17 µl of 0.025 µg/µl pUC19 DNA per class section (minimum of 34 ml per school) 3 µl Bgl 1 per class section (minimum of 6 ml per school) 15 µl reaction buffer per class section (minimum of 30 ml per school) DNA Fingerprinting

4. Supply List (cont.) 144 µl Carolina Blu Gel Stain per gel (minimum of 2 gels) 732 µl Carolina Blu Buffer Stain per gel (minimum of 2 gels) 1 bottle Carolina Blu DNA Stain 0.7 g agarose per gel (minimum of 2 gels) 70 ml 10X TBE per gel (minimum of 2 gels) 40 ml blue migration dye for each student group 8 microcentrifuge tubes for each student group Fingerprinting instructions for each student *Please send the unused pipette tips/boxes, 10X TBE bottles (rinsed out) and Carolina Blu DNA Stain back to the Office of Biotechnology when finished. DNA Fingerprinting

5. Preparation of the Student Materials • The supplies can best be provided to the class in groups of five students. • The DNA samples should be refrigerated until class time. • It is too expensive for the Office of Biotechnology to provide DNA for every student in a class. The office will provide enough DNA, restriction endonuclease, and reaction buffer for a minimum of two groups of five students or a maximum of one group of five students in every class section, whichever is greater. DNA Fingerprinting

6. DNA FingerprintingPreparation of the Student Materials(cont.) • For the other student groups, use distilled water to replace the DNA, restriction endonuclease, and reaction buffer. • Every group of students should be provided with the blue migration dye. DNA Fingerprinting

7. DNA Fingerprinting DNA and Enzyme Lab Preparation

8. DNA and Enzyme PrepStep 1 • For each group of students label 7-1.5 ml tubes "C,1,2,3,4, N and D" with a permanent felt pen. • Tube "N" will contain the enzyme mixture. • Tube "D" will contain the migration dye. DNA Fingerprinting

9. DNA and Enzyme PrepStep 2 • Pipette 17 µl of a 0.025 µg/µl concentration of pBR322 DNA into the tube labeled "C." DNA Fingerprinting

10. DNA and Enzyme PrepStep 3 • In the tube labeled "1" pipette 17 µl of 0.15 µg/µl concentration of lambda DNA. DNA Fingerprinting

11. DNA and Enzyme PrepStep 4 • In the tube labeled "2" pipette 17 µl of 0.075 µg/µl concentration of Ad-2 DNA. DNA Fingerprinting

12. DNA and Enzyme PrepStep 5 • In the tube labeled "3" pipette 17 µl of 0.025 mg/ml concentration of pBR322 DNA. DNA Fingerprinting

13. DNA and Enzyme PrepStep 6 • In the tube labeled "4" pipette 17 µl of 0.025 µg/µl concentration of pUC19 DNA. DNA Fingerprinting

14. DNA and Enzyme PrepStep 7 • In the microcentrifuge tube (1.5 ml) labeled "N," pipette 15 µl of reaction buffer and 3 µl Bgl 1. DNA Fingerprinting

15. DNA and Enzyme PrepStep 8 • In a microcentrifuge tube (1.5 ml) labeled "D," pipette 40 µl of blue migration dye. DNA Fingerprinting