/n applied biosystems by Thermo Fisher Scientific GeneMapperTM ID-X 1.6 Sample Name AM-1 AM-1 LAM-1 D3S1358 80 160 360 270- 180- 90- 0- 15 LAM-1 210- 140 70 0- 60- 828 180- 120 80 AMEL D8S1179 Χ 160 vWA D21S11 Project: 290224A D16S539 CSF1PO TPOX 240 320 400 480 240 11 D18S51 28 30 14 320 Penta E 400 480 D2S441 D19S433 TH01 FGA 80 160 240 320 400 480 0- 11 13 9.3 14 22 23 D22S1045 80 D5S818 160 D13S317 D7S820 D6S1043 240 320 400 480 150 100- 50- 0- 15 1 16 Thu Feb 29,2024 04:01PM, GMT Printed by: gmidx Page 1 of 2 applied biosystems by Thermo Fisher Scientific GeneMapperTM ID-X 1.6 Sample Name | AM-1 Project: 290224A D10S1248 D1S1656 D12S391 D2S1338 Penta D 80 160 240 320 400 480 120- 80- 40- 0- 23 25 thereum Thu Feb 29,2024 04:01PM, GMT Printed by: gmidx Page 2 of 2/n applied biosystems by Thermo Fisher Scientific GeneMapperTM ID-X 1.6 Sample Name | AM-1 LAM-1 LAM-1 LAM-1 5100 3400- 1700 Project: 280224A AMEL D3S1358 D1S1656 D2S441 D10S1248 80 160 240 D13S317 320 Penta E 400 480 0- Χ 15 14 13 12 14 17 Y 17 16 14 14 6000- 4000- 2000- D16S539 D18S51 D2S1338 CSF1PO Penta D 80 160 240 320 400 480 0 11 14 23 11 9 12 12 25 9000 6000 3000 TH01 80 vWA 160 D21S11 D7S820 D5S818 TPOX DYS391 240 320 400 480 0 9.3 15 28 16 30 10 11 11 10 10 12 D8S1179 80 D12S391 160 D19S433 FGA D22S1045 240 320 400 480 1200 800- 400 0 21 25 13 22 15 14 23 16 Thu Feb 29,2024 11:51AM, GMT Printed by: gmidx Page 1 of 1/n a.r.u. MOD00008792 - Introduction to Forensic Genetics Practical 3 - DIRECT PCR (Live Brief Practical) During this practical you will be working in pairs. Each pair will be provided with the two buccal swabs to be used for two Direct PCR. The two reactions are Prep-n-Go and VeriFiler™ Plus DNA profiling kit (PART A) and SwabSolution and PowerPlex® Fusion DNA profiling kit (PART B). The aim of the practical is to compare the results obtained from the two reaction and see if there are any significance. This practical is carried out as part of the Live Brief with AngliaDNA and it is related to E010 - Comp 2 assessment. Details of this assessment can be found on the relevant canvas site. PART A - Prep-n-Go and VeriFliler™ Plus PCR 1. Sample Preparation using Prep'n'Go Buffer 1.1. Pre-heat the heat block to 90°C. 1.2. Wearing gloves, carefully open one of the sterile swab container. Do NOT touch the sterile swab. 1.3. Cut the head of the swab into a 1.5mL microcentrifuge tube. 1.4. Add 400 μL Prep-n-Go buffer to the 1.5mL microcentrifuge tube containing the swab head and vortex for 30 second. 1.5. Place the tube on the heating block (at 90°C) for 20 minutes. (Note: While waiting you can prepare the VeriFiler Plus reaction as described in section 2). 1.6. After the time interval, remove the tube from the heating block and allow to cool down at room temperature to 15 minutes. 1.7. Quickly centrifuge the 1.5mL microcentrifuge tube with the swab head to bring down any droplets which condensed underneath the lid of the tube. 1.8. Use the solution in the 1.5mL microcentrifuge for the amplification with VeriFliler™ Plus (as described in section 2 of this protocol). 2. PCR using the VeriFliler™ Plus. Note: In this part of the procedure, you will be preparing the PCR for the buccal swab. The PCR positive and negative controls will be prepared by demonstrators and run together with your samples. 2.1. Label a 0.2mL PCR tube with your DNA and PCR number. For example for DNA sample 1 and VeriFiler PCR reaction this will be 1VF. 2.2. In the tube add the reagents listed in Table 4. a.r.u. MOD00008792 - Introduction to Forensic Genetics Table 4 - Reagents needed for the preparation of the VeriFliler™ Plus Master Mix Reagent Water (deionised and sterile) VeriFliler™ Plus Master Mix VeriFliler™ Plus Primer Mix DNA (from Sec 1) Total volume Volume (per sample) 3.0 μL 10.0 ML 10.0 ML 2.0 μL 25.0 μL Tick if added. 2.3. Mix the solution by pipetting the solution up and down. 2.4. Make sure that the 0.2 mL PCR tube is closed properly and place on PCR instrument. 2.5. Run the PCR reaction on the PCR instrument using the conditions outlined in the Table 5 Table 5 - Table showing the PCR conditions for the PowerPlex VeriFliler™ Plus. Thermocycler setting Initial denaturation Denaturation Annealing Extension Final extension Hold Cycles Time Temperature 1 1 min 95 °C 3 sec 94 °C 16 sec 59 °C 26 29 sec 65 °C 1 5 min 60 °C 1 ∞ 4ºC PART B - SwabSolution and PowerPlex® Fusion DNA profiling kit. 3. Sample Preparation using SwabSolution 3.1. Pre-heat the heat block to 70°C. 3.2. Wearing gloves, carefully open the second sterile swab container. Do NOT touch the sterile swab. 3.3. Cut the head of the swab into a 1.5mL microcentrifuge tube. 3.4. Add 1 mL of SwabSolution Reagent to the 1.5mL microcentrifuge tube and vortex for 30 seconds. 3.5. Place the 1.5 mL microcentrifuge tube with the buccal swab and SwabSolution reagent on the heating block (at 70°C) for 30 minutes. (Note: While waiting you can prepare the PowerPlex Fusion reaction as described in section 4). 3.6. After the time interval, remove the tube from the heating block and allow to cool down at room temperature for 15 minutes. a.r.u. MOD00008792 - Introduction to Forensic Genetics 3.7. Quickly centrifuge the 1.5mL microcentrifuge tube with the swab head to bring down any droplets which condensed underneath the lid of the tube. 3.8. Use the solution in the 1.5mL microcentrifuge for the amplification with PowerPlex Fusion (as described in section 4 of this protocol). 4. Sample Preparation of PCR using the PowerPlex Fusion kit. Note: In this part of the procedure, you will be preparing the PCR for the buccal swab. The PCR positive and negative controls will be prepared by demonstrators and run together with your samples. 4.1. Label a 0.2mL PCR tube with your DNA and PCR number. For example for DNA sample 1 and PowerPlex Fusion PCR reaction this will be 1PPF. 4.2. In the tube add the reagents listed in Table 6. - Table 6 – Reagents need for the preparation of the PowerPlex Fusion Master Mix Reagent Tick if added. Water (deionised and sterile) PowerPlex Fusion Master Mix PowerPlex Fusion Primer Set DNA (from Sec 3) Total volume Volume (per sample) 13.0 ML 5.0 μL 5.0 μL 2.0 με 25 μL 4.3. Mix the solution by pipetting the solution up and down. 4.4. Make sure that the 0.2 mL PCR tube is closed properly and place on PCR instrument. 4.5. Run the PCR reaction on the PCR instrument using the conditions outlined in the Table 7. Table 7: Table showing the PCR conditions of the PowerPlex Fusion kit. Thermocycler setting Initial denaturation Denaturation Annealing Extension Final extension Hold Cycles Time Temperature 1 1 min 96 °C 10 sec 94 °C 1 min 59 °C 27 30 sec 72 °C 1 20 min 60 °C 1 ∞ 4ºC After the PCRs with the VeriFliler™ Plus and PowerPlex Fusion, the samples will be run on a Capillary Electrophoresis instruments and results will be provided later.