1. H OOSIER DADDY ? Y OU ARE NOT THE FATHER ! Ward’s 36-8551 Paternity testing lab activity Washington H.S. Kasi Bolden

2. M ATERIALS INCLUDED IN KIT Agarose 5x TBE running buffer DNA stain Amber bottle Gel staining trays Biohazard bag DNA samples (mother, child 2 alleged fathers)

3. M ATERIALS NEEDED Agarose electrophoresis chambers Power supplies Water or microwave Micropipets with tips Apron, lab markers & goggles UV transilluminator UV goggles

4. O BJECTIVE Learn the process of agarose gel electrophoresis Perform the electrophoresis procedure Identify the most probable DNA match between two alleged fathers

5. P RIOR K NOWLEDGE DNA fragment length DNA fingerprinting technology vs. paternity testing DNA molecules found in a human cell

6. F UN FACTS ABOUT DNA DNA testing was used to prove that Thomas Jefferson was in fact the biological father of at least on child of his slave, Sally Hemmings. Electrophoresis is derived from the Greek electros, meaning electricity and phoros, meaning “to carry across” Agarose is highly purified for of agar, which is material extracted from seaweed. Gel electrophoresis issues in genetic testing, paternity analysis, forensics, conservation biology, animal behavior, taxonomy and evolutionary biology

7. F UN F ACTS CONTINUED The first time DNA was used as evidence in a trial was in 1985. The first time DNA evidence actually sent someone to jail was in 1988. If all of the DNA molecules found in a single human cell were lined up end-to end, they would reach a distance a of about two meters. Yet, all of these molecules are packed into a nucleus 10 millionths of a meter in diameter

8. A NNETTE G ORDON -R EED DISCUSSES ' PASSING ' AND THE EFFECT IT HAD ON LIVES AND FAMILIES. T HE INTERVIEW WAS RECORDED IN O CTOBER 2008 AT M ONTICELLO ' S R OBERT H. S MITH I NTERNATIONAL C ENTER FOR J EFFERSON S TUDIES. http://www.youtube.com/watch?v=CHWtkml0suc &feature=PlayList&p=C63B8B73DBE0CB39&pl aynext_from=PL&playnext=1&index=23

9. T IME Day 1 Casting gels and diluting buffer: 30 min. Day 2 Loading and running gel: 60 min. Staining and analyzing gel: 60min

10. S CENARIO Two men claim to be the biological father of child. The courts have obtained DNA samples from the mother, child and the two alleged fathers. As the director of a genetic testing laboratory, you will analyze the sample using the DNA fingerprinting technique to help settle the paternity dispute.

11. D AY 1:C ASTING AN AGAROSE GEL 1. Add 280mL of distilled water to 70mL of 5X TBE concentrated to obtain 350mL of 1X buffer. 2. Melt the agarose gel using a boiling water bath or a microwave. 3. Place the comb on the far side of the casting trays. 4. Pour approximately 25 mL melted agarose into the tray, until it reaches a depth of about 3 mm (NOTE: The 1X TBE buffer will be added after the gel has formed.) 5. Allow the gel to solidify for approximately 20-30 minutes. Do not disturb 6. After the gel solidified, carefully remove the comb.

12. D AY 2: L OAD GEL 1. Place the wells closer to the negative ( black) electrode. 2. Add the 1X TBE buffer until the gel it completely submerge. 3. Load each DNA sample; 10µL of each DNA sample. 4. #1 Mother in, #2 Child, #3 alleged Father1 & #4 alleged Father 2

13. D AY 2 PROCEDURE 5. Allow gel to run for approx. 30mins @125V or until the dye migrates towards the red electrode. You should see bubbles. 6. Carefully remove the gel, on the casting tray, from the electrophoresis chamber. Pour approx. 100mL warm dilute. (If the lab period does not allow time to stain and destain the gel, place the gel in a resealable bag and add 1-2mL of 1X TBE buffer and refrigerate the gel until the next lab period.) 7. Accurately sketch the bands you see on the blank gel in the analysis section.

14. DISCUSSION Would you modify this lab? If so how? What level of science would you do this lab? Could you use this as a cross curricular activity? Thumbs up or thumbs down

15. F INALE Based on the autoradiogram the child will have inherited bands from the mother and the alleged father.