AccuID TM _V1. Bone DNA Preparation Protocol. SNP based New Human Identification Technology. Protocol Version

AccuID TM Bone Preparation Protocol 2 Assay Overview Ablation Decalcification Chopping Lysis PCI Enrichment Purification Figure 1. Overview of the AccuID TM Bone DNA Preparation

AccuID TM Bone Preparation Protocol 3 Reagents, Equipments and Other Materials Required Assay Stage Reagents Equipments Ablation - Jouan MSC 12 Class II Microbiological Safety Cabinet: Thermo Fisher Scientific FM-01-3-Dust Collectors, Sky Blue C: Frontier Dental Industries. Hand drill: Marathon HANDY702, P/N 7200 Carbide drill bit, Midwest, P/N 385204 3M Half Face-piece Reusable Respirator Assembly: 3M, P/N 6291 50ml Centrifuge tube: Corning, P/N 430290. Decalcification 0.5M EDTA, ph 8.0: Bioneer Molecular Biology Grade Water: Thermo Scientific, P/N SH30538.02 Chopping Agilent DNA 1000 Kit, P/N 5067-1504 Agilent High Sensitivity DNA Kit, P/N 5067-4627 Lysis 1M Tris-HCl, ph 8.0-1ml: Bioneer, P/N C- 9006 5M NaCl 2ml: Life Technologies, P/N 24740011 SDS: Sigma, P/N 71736-500ML Proteinase K: MBiotech, P/N 19910 1M DTT: Sigma, P/N 43815-25G Molecular Biology Grade Water: Thermo Scientific, P/N SH30538.02 PCI (Phenol/Chlorofom/Isoamyl Alcohol) Phenol:Chloroform:Isoamylalcohol (25:24:1) for DNA purification, 500 ml: Bioneer, P/N C-9017 Enrichment MinElute PCR Purification Kit (50): Qiagen, P/N 28004 Microcon YM-100: Millipore Purification MinElute PCR Purification Kit (50): Qiagen, P/N 28004 Shaking Incubator + Wrie rack 660X520: Jeiotech, P/N SI-600R Filter Paper: Hyundai, P/N HD51-090 Scalpel: Paragon, P/N 0891006 Hybridization oven: Agilent Technologies, P/N 1013AG-2 Allegra X-15R Centrifuge: Beckman Coulter, P/N 392932 Sorvall Legend Micro 17 Microcentrifuge: Thermo Scientific, P/N 75002430 Vortex Mixer IKA-Model MS2- S8/MS2-S9 Sorvall Legend Micro 17 Microcentrifuge: Thermo Scientific, P/N 75002430

AccuID TM Bone Preparation Protocol 4 STAGE 1: Ablation Ablation is performed inside a dust collector in a fume hood. 1. Using a hand drill with a appropriate drill bit size for each bone, remove damaged surface of the bone. 2. Change drill bit for each bone. 3. Change gloves for each bone. STAGE 2: Decalcification 1. Immerse the bone into 40 ml EDTA buffer (0.5M, ph 8.0) in a 50 ml conical tube. 2. Fix the tubes in a shaking incubation and run for 2 weeks at room temperature. 3. Change the buffer 3 times a week during the incubation. Pour out the old buffer and wash with. by loading. and inverting the tube a few times and pouring. back out of the tube. Then, load a new buffer. STAGE 3: Chopping 1. Dry up the moisture of the decalcified bone using a filter paper. Place a filter paper on top of a paper towel. Place a decalcified bone on top of them and then roll it with filter paper and paper towel with enough pressure in order to dry the bone as much as possible. Perform it few times until enough moisture has dried from the bone. 2. Using a scalpel, first slice the surface of the bone as thinly as possible then chop the sliced pieces into as many tiny pieces as possible. The smaller the chopped pieces, the easier it is to lyse. 3. Put the chopped bone into a 15 ml conical tube. NOTE - For each bone, a minimum amount of 3 grams of chopped sample is needed. For each prep, 1.5 grams is needed twice. NOTE - 1.5 g of chopped bone fills up the 15 ml conical tube up to around 5 ml.

AccuID TM Bone Preparation Protocol 5 STAGE 4: Lysis 1. Prepare a lysis buffer (Table 1) by using the following tables (Table 2-5). Table 1. Preparation of Lysis Buffer (based on 10 ml volume) 1X TEN buffer 7.5 ml SDS (10%) 0.75 ml Proteinase K (10mg/ml) 1 ml 1M DTT 0.75 ml Table 2. Preparation of 1X TEN Buffer (based on 100 ml volume) 1M Tris-HCl(pH 8.0) 1 ml 5M NaCl 2 ml 0.5M EDTA 2 ml 95 ml Table 3. Preparation of 10% SDS (based on 100 ml volume) SDS 10 g Up to 100 ml Table 4. Preparation of Proteinase K (10 mg/ml) (based on 100 ml volume) Proteinase K 1 g Up to 100 ml Table 5. Preparation of 1M DTT (based on 100 ml volume) DTT 15.42 g Up to 100 ml 2. Load the lysis buffer into the 15 ml tube containing the chopped bone so that it just fills up the bone 3. Run the tubes in a 56 inverting incubator for 2-3 hours or until the bone completely dissolves.

AccuID TM Bone Preparation Protocol 6 STAGE 5: PCI (Phenol/Chlorofom/Isoamyl Alcohol) 1. Completely fill the lysate tube with phenol up to 16 ml. NOTE - It is customary to fill an equal amount of phenol as that of the lysate, but since the volume of lysate varies between samples, phenol is filled completely for the centrifugal balance purpose. 2. Invert the mix with hand for 1-2 minutes so that phenol and lysate mixes completely. 3. Centrifuge at 13,000 rpm for 10 minutes at 4. 4. Carefully collect the supernatant into a new 15 ml tube and completely fill the tube with phenol. Try not to collect the layer between the supernatant and sediment. 5. Invert the mix completely. 6. Centrifuge at 13,000 rpm for 10 minutes at 4. 7. Carefully collect the supernatant into a new 15 ml tube. Do not to collect the white layer between the supernatant and sediment. STAGE 6: Enrichment NOTE - Use 1 Microcon for 1 ml of supernatant. 4-5 Microcons are needed for each sample. 1. Load 500 µl supernatant into each Microcon. 2. Centrifuge at 4000 rpm for 40 minutes or until the liquid completely passes through the membrane. Do not centrifuge at a high speed for a possible membrane damage. 3. Discard the flow-through and check the speed at which the sample passes through the membrane. 4. Load the rest of the supernatant (450~500 µl) into the Microcon. 5. Centrifuge at 4000 rpm for more than 40 minutes. Second centrifugation requires more time for membrane pass. 6. Discard the flow-through. 7. Load 100 µl Qiagen EB Buffer and let it rest at room temperature for 5 minutes. 8. Centrifuge at 4000 rpm for 5 minutes.

AccuID TM Bone Preparation Protocol 7 STAGE 7: Purification 1. Remove the Microcon and load PB 5 times the volume of EB Buffer. Mildly vortex and spin down. 2. Load the mix into a new Qiagen column and centrifuge at 13,000 rpm for 1 minute. 3. Discard the flow-through. 4. Repeat steps 2 and 3 onto the same column for the tubes of the same sample. 5. Load 700 µl PE and centrifuge at 13,000 rpm for 1 minute. 6. Discard the flow-through. 7. Carefully load 50 µl EB into the center of the column. 8. Let it rest at room temperature for 5 minutes. 9. Centrifuge at 13,000 rpm for 1 minute. 10. Store the flow-through.