1. Biotechnology Explorer™ | explorer.bio-rad.com 1 Biomedical Science Activity 1.3.1 DNA Detectives Damon_Tighe@Bio-Rad.com

2. Biotechnology Explorer™ | explorer.bio-rad.com 2 PLTW Biomedical Science 1.Show prep 1.Recommendations 1.Further dilute Enzymes so adding 5ul not 1ul 2.Stopping points 2.Show procedure 1.Fast Blast 2.UView

3. Biotechnology Explorer™ | explorer.bio-rad.com 3 Goal  Students receive DNA from bones and through restriction digestion and gel electrophoresis see if they match one of two known missing persons

4. Biotechnology Explorer™ | explorer.bio-rad.com 4 Preparation Day 1Day 2Day 3Day 4 Teacher Prep for Restriction Digestion lab & TAE Student Lab Restriction Digestion & Gel Pouring Student Lab Gel Electrophoresis Student Lab Gel analysis Order kit up to 1 year out

5. Biotechnology Explorer™ | explorer.bio-rad.com 5 Preparation  Order kit 1660030EDU  Receive kit and store reagent bag at -20C If you don’t have -20C freezer and you want to store biologicals long term you can freeze the items in a block of ice, which prevents freeze/thaw cycles and ultimately ice crystals from “knifing” the biological material to bits. A styrofoam cooler in the freezer could also work. Bio-Rad Guarantees kits for 1 year from date received

6. Biotechnology Explorer™ | explorer.bio-rad.com 6 Preparation – “bone sample” DNA digests  Add 220µl of sterile water to Suspect 1vial  Add 200µl of sterile water to Suspect 2 vial  Mix and allow to sit for 15 minutes

7. Biotechnology Explorer™ | explorer.bio-rad.com 7 Preparation – “bone sample” DNA digests Reaction1 Reaction2 Sterile Water 40.5 µl DNA Suspect1 45 µl HindIII4.5 µl Total90µl Sterile Water 40.5 µl DNA Suspect1 45 µl PstI4.5 µl Total90µl Reaction 1Reaction 2 37C, 45 minutes Or

8. Biotechnology Explorer™ | explorer.bio-rad.com 8 Preparation – aliquot Bone DNA digestions Reaction1 Bone HindIII 10µl X 8 Reaction2 Bone PstI 10µl X 8

9. Biotechnology Explorer™ | explorer.bio-rad.com 9 Preparation – Lambda DNA Ladder 100µl Lambda HindIII digest DNA 20µl Sample Loading *65C, 5 minutes lambda *best done day of gel electrophoresis Dye

10. Biotechnology Explorer™ | explorer.bio-rad.com 10 Preparation – aliquoting reagents (PLTW) Missing 1 Missing 2 Marker HindIII Pst1 Sterile water Loading Dye X 8 of each 11µl 12µl 3µl 50µl 30µl Lambda HindIII Pst1

11. Biotechnology Explorer™ | explorer.bio-rad.com 11 Preparation – aliquoting reagents Damon’s Recommendation Missing 1 Missing 2 Marker HindIII Pst1 Loading Dye X 8 of each 11µl 12µl 3µl + 12µl water 30µl Lambda HindIII Pst1 3µl + 12µl water

12. Biotechnology Explorer™ | explorer.bio-rad.com 12 Preparation – storage recommendation Missing 1 Missing 2 Marker HindIII Pst1 Sterile water Bone HindIII Bone PstI Loading Dye Add to foam float, since these will all be used 1 st lab day to do the digestion of DNA Keep these for the Gel Electrophoresis day

13. Biotechnology Explorer™ | explorer.bio-rad.com 13 Preparation – TAE  Make 1x TAE from 50x TAE  You’ll need ~ 3L of 1x TAE for the lab: –60mL 50X TAE –2,940 mL distilled water –OR to a 1Gallon (3.78L) of Distilled water Add ~76.5mL 50X TAE

14. Biotechnology Explorer™ | explorer.bio-rad.com 14 Preparation – 1 X Fast Blast Stain  Add 1mL of 500x Fast Blast Stain to 500mL of Distilled Water. Drinking water also works.

15. Biotechnology Explorer™ | explorer.bio-rad.com 15 Preparation – Lab day 1 workstation (8) Missing 1 Missing 2 HindIII Pst1 Sterile water 4 clean microcentrifuge tubes *

16. Biotechnology Explorer™ | explorer.bio-rad.com 16  Bones police recovered and worked by your classmates have helped narrow down the field to two missing persons. You will be using restriction digestion and control samples from the two missing persons to determine whose remnants were found. Student Lab - Scenario

17. Biotechnology Explorer™ | explorer.bio-rad.com 17 Student Lab - Scenario  Missing Person #1- Jack Matthews, 21, a senior at the local university, left his apartment at approximately 10 am headed for his Biotechnology class. When Jack did not show up for class, his lab partner alerted his roommate; when Jack did not show up at the apartment that night, his roommate called the police and Jack’s parents. Three months have passed and no one has heard from Jack. Two witnesses remember seeing him on the path near the park on the day he disappeared, but the police have no other leads.

18. Biotechnology Explorer™ | explorer.bio-rad.com 18  Missing Person #2- Robert Jones, 24, a newly married insurance salesman, has been missing for almost six months. On the day he disappeared, he spoke to his wife on the phone at approximately 4pm. He told her that he was going to stop at the gym for a workout before coming home; however, the gym has no record of his signing in that day. His wife admits that they were having trouble adjusting to their new life, but she never thought he would simply up and run away. Student Lab - Scenario

19. Biotechnology Explorer™ | explorer.bio-rad.com 19 Sterile water  Label 4 Microcentrifuge tubes 1-4  In notebook, record what will be in tubes  *Add 4µL sterile water to each tube Student Lab - Procedure Day1 1 2 3 4 Tube 1 – Missing Person #1 cut with HindIII enzyme Tube 2 – Missing Person #2 cut with HindIII enzyme Tube 3 – Missing Person #1 cut with PstI enzyme Tube 4 – Missing Person #2 cut with PstI enzyme * don’t add if you are using Damon’s recommendation of pre-diluting enzymes during the prep

20. Biotechnology Explorer™ | explorer.bio-rad.com 20  Add DNA and then *Enzymes to tubes  Close caps, flick to mix and shake down each tube Student Lab - Procedure Day1 1 2 3 4 Tube 1 – 5 µl Missing Person #1 DNA + 1 µl HindIII enzyme Tube 2 – 5 µl Missing Person #2 DNA + 1 µl HindIII enzyme Tube 3 – 5 µl Missing Person #1 DNA + 1 µl PstI enzyme Tube 4 – 5 µl Missing Person #2 DNA + 1 µl PstI enzyme * If following Damon’s recommendation of pre-diluting enzyme, pipette 5ul of each enzyme above

21. Biotechnology Explorer™ | explorer.bio-rad.com 21  Place tubes in a float and put in waterbath for 30 minutes and 37C  Store at 4C until next lab day Student Lab - Procedure Day1

22. Biotechnology Explorer™ | explorer.bio-rad.com 22  1% gel = 1g agarose + ~99g (mL) 1x TAE  Each 7x10cm tray needs ~50mL gel  Tape or bumper your trays and add combs  In a glass container with 2x head space combine 1X TAE and agarose. Shake to mix.  Microwave in glass container with 2 x head space with a napkin over the top for 30 seconds.  Pull out, gently shake and repeat until solution is COMPLETELY clear.  Pour into tray until gel is half way up teeth of comb Student Lab – Pour 1% Agarose gels Day1

23. Biotechnology Explorer™ | explorer.bio-rad.com 23 Preparation – Lab day2 Gel electrophoresis 1 2 3 4 Loading Dye Samples Gel powersupply Gel box Marker

24. Biotechnology Explorer™ | explorer.bio-rad.com 24  Heat Marker for 2 minutes at 95C  Retrieve DNA digest samples 1-4 from 4C  Add 2µl Loading Dye to tubes 1-4, flick to mix Student Lab – Gel Electrophoresis Day2 Loading Dye Marker

25. Biotechnology Explorer™ | explorer.bio-rad.com 25  Set up your gel boxes –Place gels in box –Cover with 1X TAE  Load your gels, record your loading order in notebook  Run 30 minutes at 125V Student Lab – Gel Electrophoresis Day2 Lane 1: 10 µl Standard DNA Marker Lane 2: 10 µl DNA from Bone cut with HindIII Lane 3: 10 µl Tube 1/ Missing Person #1/HindIII Lane 4: 10 µl Tube 2/Missing Person #2/HindIII Lane 5: 10 µl DNA from Bone digested with PstI Lane 6: 10 µl Tube 3/Missing Person #1/PstI Lane 7: 10 µl Tube 4/Missing Person #2/PstI Can run 20 minutes at 200V if you use 0.25X TAE running buffer

26. Biotechnology Explorer™ | explorer.bio-rad.com 26  Remove gels from trays and place in tupperware container.  Cover with 1x Fast Blast stain and let sit overnight Student Lab – Gel Electrophoresis Day2

27. Biotechnology Explorer™ | explorer.bio-rad.com 27  Pour Fast Blast from gels down the sink  Fill tupperware with warm water for 1-2 minutes  Place gel on white background and photograph Student Lab – Document Gel Day3

28. Biotechnology Explorer™ | explorer.bio-rad.com 28 Student Lab – Analyze Gel Day3 marker BoneMP1MP2BoneMP1MP2 HindIIIPstI 23,000 9,400 6,500 4,400 2,300 2,000 bases

29. Biotechnology Explorer™ | explorer.bio-rad.com 29 Student Lab – Analyze Gel – Got math? Day3 marker BoneMP1MP2BoneMP1MP2 HindIIIPstI 23,000 9,400 6,500 4,400 2,300 2,000 bases 1.Measure distance from well to bands in the marker 2.Plot distance versus basepairs 3.Find a line of best fit 4.Use the equation of a line to solve for the size of the other bands 5.Compare the samples based upon basepairs to solve mystery basepairs distance basepairs distance

30. Biotechnology Explorer™ | explorer.bio-rad.com 30 What did we use today? Bio-Rad’s DNA Detectives

31. Biotechnology Explorer™ | explorer.bio-rad.com 31  Things I need to bring –Gel caster –UV Transilluminator –Microplate reader –Tupperware x 8 - Green Racks 8