Collect Buccal Cells

1. Collect Buccal Cells

2. PCR Polymerase Chain Reaction DNA/gene amplification

3. Chromosome 8 and Alu element Alu + form Alu - form

4. Preparing an Agarose Gel for Electrophoresis

5. Reagents and Supplies for Gel Electrophoresis • Scale - for weighing agarose • Spatula – for scooping agarose powder • Flask or beaker - for melting agarose • Graduated cylinder for measuring buffer • Microwave or hot plate and large beaker to boil water • Agarose • Buffer • Gel tray(s) and comb(s) • Gel box(es) • Power supply • DNA Staining solution • Photo doc. system

6. Reagents and Supplies for Gel Electrophoresis Continued • Your DNA Sample • Loading dye- for help with loading the gel • DNA Ladder or Marker • 20uL Pipettes • Tips • Gloves • Waste container

7. Terms • Agarose is a polysaccharide (carbohydrate) polymer material, generally extracted from seaweed. • Gel electrophoresis is a method that uses an electrical current and a gel matrix to separate molecules like DNA and proteins. • Buffer a solution containing either a weak acid and its salt or a weak base and its salt, which is resistant to changes in pH.

8. Terms continued • Ethidium Bromide a fluorescent chemical that intercalates between base pairs in a double stranded DNA molecule. Commonly used to detect DNA following gel electrophoresis. • DNA Ladder consists of known DNA sizes used to determine the size of an unknown DNA sample. The DNA ladder usually contains regularly spaced sized samples which when run on an agarose gel looks like a "ladder". • Power Supply a source of electric current

9. Percent (%) Solutions • Percent Weight/Volume (w/v) solutions are prepared by dissolving a solute usually a solid material into 100 mL of a solution such as water, buffer, DMSO, etc. Some solutes are not easily dissolved in solvents so you may need to stir, heat, sonicate or vortex the solution until it dissolves. • Other types of % solutions are Weight/Weight (W/W) and Volume/Volume (V/V) solutions

10. Preparing a 2% W/V Agarose Gel • 2% W/V agarose = 2 grams agarose dissolved in 100 milliliters (mL) of Buffer • For our purposes each gel tray requires 25 mLs of gel • To prepare 25 ml of a 2% agarose gel 2 grams = X grams 100mls 25 mls X = 0.5 grams agarose • Weight 0.5 grams agarose and dissolve in 25 mls buffer

11. Directions for preparing and loading Gel • Weigh out amount of agarose, measure volume of buffer needed and place in flask or beaker • Microwave or use hot plate to dissolve agarose note: do not let melted agarose sit for too long, it will solidify in the beaker! • Place comb into casting tray, raise dams on both ends of tray and pour gel into casting tray • Allow gel to solidify • While gel is solidifying add 4ul of loading dye to your DNA in your PCR tube • Pour buffer into gel box (~250 mL)

12. Directions for preparing, loading and running Gel continued After gel solidifies: • Carefully remove comb from gel, put dams down on both ends of the gel tray • Place gel tray into gel box with buffer • Load 10uL of the Marker usually in lane 1 of each gel and your DNA sample 20uL per well. Once everyone has loaded their DNA sample plug red electrode to red and black electrode to black on power supply, make sure the power is turned off on power supply before connecting electrodes! • Adjust voltage to 125-135 volts allow gel to run at least 15 minutes, 30 minutes is ideal

13. Directions continued • After gel has run for at least 15 minutes, turn off power supply • Unplug electrodes • Place gel into tray and pour gel staining solution over gel. The entire gel should be covered with solution • Place tray on rocker for 15 to 30 minutes • Take gel in tray to photodoc station • Place gel on trans illuminator and take picture • Record your genotype in your lab notebook

14. Gel Electrophoresis

15. Gel Electrophoresis

16. Chromosome and Alu element Alu + form Alu - form

17. Alu Alleleon Chomosome 8