PCR Lab Classroom Day One

Updated Amgen Lab 8: Genomic DNA Extraction from Buccal Epithelial Cells and Amplification of the t A Locus using the ol!merase Chain "eaction

Materials
Sterile pipette tips Boiling ater bath #igh$spee% &entrifuge Chelex tube: 0.2 mL of 10% Chelex solution !C" tubes !C" 'a&hine ()hermal Cy&ler* $ &ollege

Day ONE: Collecting your DNA sample for PCR thermocycler

1. Obtain a Chelex tube. +ote that this tube is i%entifie% ith a number. "e&or% this number in your noteboo,. Only you ill ,no this anonymous &o%e. 2. )o &olle&t bu&&al epithelial &ells- use a sterile toothpi&, or sterile pipette tip to gently s&rape the inside of both &hee,s. S&rape ell so that you get lots of &ells. #o e.er- this pro&e%ure shoul% be non$in.asi.e so %on/t %ra bloo%. 0. )ransfer the &ells that you ha.e remo.e% from the toothpi&,1pipette tip to the Chelex tube. 2igorously t irl the toothpi&,1tip in the Chelex resin to ,no&, the &ells off toothpi&,1 tip. )his step is important3 you ant to get as many &ells off the toothpi&,1pipette tip an% into the Chelex tube as possible. 4. Close the Chelex tube tightly. !la&e your tube in the ra&, hi&h ill be pla&e% in the boiling ater bath or pla&e your tube %ire&tly into the 100C hot blo&,. Boil or heat the &ells for 10 minutes. )his heating ill lyse the &ells an% help to %estroy some of the nu&leases- hi&h &an %egra%e the D+5. 6. 7se the high$spee% &entrifuge set to 810-000 rpms to spin %o n the Chelex an% &ell %ebris for 6 minutes. 9. 7sing the !$20 pipette an% a &lean pipette tip- &arefully remo.e 6 L of supernatant ( hi&h &ontains your genomi& D+5* an% pla&e it into a &lean !C" tube. 5.oi% aspirating Chelex bea%s as this ill inhibit the %o nstream !C" pro&e%ure. Label the cap an si e of this !C" tube ith your personalanonymous &o%e. :. !la&e your !C" tube an% Chelex tube into the ra&,s at the front of the room. ;our genomi& D+5 sample &an be store% in the free<er at $20=C until your tea&her is rea%y to run the !C" rea&tion.