D3S1358 Analysis

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Appendix 1
Analysis of D1S80 and D3S1358
For each subject a sterile swab was rubbed on the inside surface of the cheek before being cut and transferred into a 1.5ml Eppendorf tube before 300µl of QuickExtract™ DNA extraction was added followed by a vortex for 15 seconds. The Eppendorf tube was then heated to 65°C and incubated for 10 mins before a second phase of a 15 second vortex. Finally, the Eppendorf tube was heated at 98°C and incubated for 2 minutes before being stored on ice.
DNA Amplification via Polymerase Chain Reaction This step was performed twice in separate tubes, once for D1S80 containing the hot start Master Mix bead and once for D3S1358 containing the ready to go Master Mix bead. In an Eppendorf tube containing
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Then 5µl of DNA samples were transferred into an individual Eppendorf tube containing the master mix solution. Once completed all 15 tubes were immediately transferred to a thermal cycler.
Restriction Digestion of Amplified Polymorphic Regions MstII digestion of 15µl amplified reaction, 1µl of MstII was added to the reaction and then incubated at 37°C for 1-2 hours and this incubation period the reaction was terminated by heating the to 70°C for 5 minutes followed by rapidly cooling on ice. This was then stored at 4°C.
Preparation of Gel The 2% agarose was prepared by dissolving 2g of agarose (Sigma NA grade) in 98ml of sterile H₂0, the solution was then heated until dissolved and allowed to cool to 55°C before adding 2ml of 50X tris-acetate-EDTA (TAE) buffer and 10µl of gel red™ Nucleic acid stain. The gel was then poured into a gel tray with a 20µl slot comb already fitted. All visible air bubbles were removed and the gel allowed to set.
Sample Preparations Into each sample of restriction amplified polymorphic regions 3µl of 6x sample loading buffer was added. 10µl of ladder was added into well 1 and then 10µl of sample was loaded in the wells as described in table 2.
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