Forensic DNA Fingerprinting Teacher Lab Prep Suggested Timeline

Forensic DNA Fingerprinting: Teacher Lab Prep

Suggested Timeline Pre-lab Day before Lesson 1 Day of Lesson 1 Prior to Lesson 2 • m Teacher Prep Student Lab Lecture / Questions Click on blue and green arrows to jump to procedures Lesson 2 & Post-Lab

Forensic DNA Fingerprinting Preparation Check contents of Forensic DNA Fingerprinting Kit

Forensic DNA Fingerprinting Preparation prep Additional Equipment (not in kit)

Forensic DNA Fingerprinting Preparation prep 1. Prepare TAE Buffer Make 3 L of 1 x TAE buffer from 50 x TAE solution: 60 ml 50 x TAE + 2. 94 L water

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 2. Prepare 1% Agarose Gels 1. In a flask combine: 500 ml 1 X TAE + 5 g agarose 2. Heat with a microwave or a hot plate until solution boils and is completely clear Caution: don’t allow agarose to boil over. Solution is burn hazard (continued)

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 2. Prepare 1% Agarose Gels 3. Use lab tape to tape the end of the gel trays 4. Place 1 comb in the top of the gel tray Note: if two student groups are using the same gel, place another comb in the middle of the tray (continued)

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 2. Prepare 1% Agarose Gels 5. Once the flask is comfortable to hold, pour agarose into tray to half the depth of the comb’s teeth 6. Allow 10 minutes for gel to solidify 7. Remove comb and tape, and store gels at 4ºC until used

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 3. Rehydrate DNA Samples 1. Add 200µl of Sterile Water to each vial of DNA using a new tip for each sample 2. Let stand at room temperature for a minimum of 15 minutes Note: 37ºC for 10 minutes may help getting DNA in solution

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 4. Rehydrate Enzyme Mix 1. Add 750µl of Sterile Water to Eco. RI/Pst. I Enzyme Mix on ice Note: use enzymes within 12 hours of rehydrating

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 5. Aliquot Enzyme for student stations 1. Label 8 clear micro test tubes “ENZ” 2. Aliquot 80µl of Eco. RI/Pst. I Enzyme Mix into the micro test tubes labeled “ENZ” 3. Store at 4ºC until used

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 6. Set up water bath 1. Set water bath or dry bath to 37ºC 1 hour prior to the start of lab Note: if water bath is not available, heat water to 37 C and keep warm in styrofoam cooler or let restriction digestion take place at room temperature overnight

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 7. Set up student work stations for lab 1

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 8. Student Lab 1: Restriction Digest (continued)

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 8. Student Lab 1: Restriction Digest (continued)

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 9. Prepare DNA Loading Dye 1. Label 8 clear micro test tubes “LD” 2. Aliquot 50µl of DNA sample loading dye to tubes “LD”

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 10. Prepare Hind. III lambda DNA marker 1. Label 8 clear micro test tubes “M” 2. Add 20µl of DNA sample loading dye to Hind. III lambda (DNA marker) vial 3. Heat Hind. III lambda (DNA marker) to 65ºC 5 minutes and place on ice 4. Aliquot 15µl of Hind. III Lambda (DNA marker) to the tubes labeled “M”

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 11. Prepare Fast Blast Dilute Fast Blast DNA Stain for quick staining or overnight staining: • Quick Staining (100 X Stain): Combine 100 ml Fast Blast with 400 ml water • Overnight Staining (1 X Stain): Combine 1 ml Fast Blast with 499 ml Store Fast Blast at room temperature until used

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 12. Set up electrophoresis chambers 1. Remove tape and comb from gel 2. Place tray into gel chamber with the wells of the gel closest to the black electrodes (-) 3. Add 1 x TAE Buffer until wells are completely covered

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 13. Set up student workstations for lab 2

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 14. Student Lab 2: Gel Electrophoresis (continued)

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 14. Student Lab 2: Gel Electrophoresis Load total volume (continued)

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 14. Student Lab 2: Stain Gels Cover top of gel with stain. (continued)

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 14. Student Lab 2: Stain Gels

Forensic. DNAFingerprinting Forensic DNA Fingerprinting Preparation prep 15. Expected results