Forensic DNA Fingerprinting: Teacher Lab Prep

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

Check contents of Forensic DNA Fingerprinting Kit Forensic DNA Fingerprinting Preparation Check contents of Forensic DNA Fingerprinting Kit

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

Make 3L of 1x TAE buffer from 50x TAE solution: 60 ml 50x TAE Forensic DNA Fingerprinting Forensic DNA Fingerprinting Preparation prep 1. Prepare TAE Buffer Make 3L of 1x TAE buffer from 50x TAE solution: 60 ml 50x TAE 2.94 L water +

Caution: don’t allow agarose to boil over. Solution is burn hazard + Forensic DNA Fingerprinting Forensic DNA Fingerprinting Forensic DNA Fingerprinting Preparation prep prep 2. Prepare 1% Agarose Gels 1. In a flask combine: 500ml 1X TAE 5g 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)

3. Use lab tape to tape the end of the gel trays Forensic DNA Fingerprinting Forensic DNA Fingerprinting Forensic DNA Fingerprinting Preparation prep 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)

6. Allow 10 minutes for gel to solidify Forensic DNA Fingerprinting Forensic DNA Fingerprinting Forensic DNA Fingerprinting Forensic DNA Fingerprinting Preparation prep prep 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

2. Let stand at room temperature for a minimum of 15 minutes Forensic DNA Fingerprinting Forensic DNA Fingerprinting Forensic DNA Fingerprinting Forensic DNA Fingerprinting Preparation prep prep 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

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

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

Set water bath or dry bath to 37ºC 1 hour prior to the start of lab Forensic DNA Fingerprinting Forensic DNA Fingerprinting Forensic DNA Fingerprinting Forensic DNA Fingerprinting Preparation prep prep prep 6. Set up water bath 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 37C and keep warm in styrofoam cooler or let restriction digestion take place at room temperature overnight

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

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

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

1. Label 8 clear micro test tubes “LD” Forensic DNA Fingerprinting Forensic DNA Fingerprinting Forensic DNA Fingerprinting Forensic DNA Fingerprinting Preparation prep prep 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”

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

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

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

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

Forensic DNA Fingerprinting Forensic DNA Fingerprinting Forensic DNA Fingerprinting Preparation prep prep 14. Student Lab 2: Gel Electro- phoresis (continued)

Forensic DNA Fingerprinting Forensic DNA Fingerprinting Forensic DNA Fingerprinting Preparation prep prep 14. Student Lab 2: Gel Electro- phoresis Load total volume (continued)

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

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

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