Overview: Human Alu insertion polymorphism lab steps

1. Overview: Human Alu insertion polymorphism lab steps 1. Cheek cell (Method A) or hair sheath cell (Method B) sampling - saline (0.9% NaCl) mouth wash and cell centrifugation (cheek cells) - add protease solution (hair follicle cells) - add Chelex solution (cheek cells) - chelex is a chelating agent which traps divalent ions to prevent DNA degradation during lysis and PCR 2. DNA isolation - incubate cells 56oC for 10 min; then vortex vigorously - incubate cells at 100oC, 6 min; then vortex vigorously 3. Centrifuge samples in microcentrifuge 4. Take supernatant, transfer, add Master mix and performPolymerase chain reaction (PCR) to amplify the chromosomal PV92 region - mix 10l DNA with 20l PCR Master mix and 20l Alu PV92 primer - perform thermal cycling step using following temperature program: 95oC, 10 min 94oC, 30sec 65oC, 30sec 30 cycles 72oC, 2 min 72oC, 10 min 5. Agarose gel electrophoresis - load PCR samples onto a 1.0% EtBr-agarose gel and perform DNA electrophoresis at 100 - 125V for 30 – 45 min 6. DNA staining with Ethidium Bromide (EtBr) dye or Fast Blast™ DNA blue stain - PCR amplified DNA becomes visible 7. DNA band analysis: Determination of your PV92 genotype & Hardy-Weinberg analysisof the class results - presence of 715bp fragment indicates Chr. #16 Alu insert - 415bp PCR amplicon indicates absence of Alu insert on PV92 locus Genotypes: M - / - + / - + / + Control Alu heterozygot DNA size (bp) 1000 715bp 700 500 415bp 400 300 PCR amplicon 200 100 Agarose gel + Blue stain