Lab 11: Morning Lark or Night Owl? Circadian Clock Internal - PowerPoint PPT Presentation

NOTEBOOK Lab 11: Morning Lark or Night Owl?

Circadian Clock • Internal timekeeper that regulates the body’s physiology and behavior on a cycle that repeats each day • Other animals, plants, fungi and unicellular organisms have circadian clocks to help transition between light and darkness

NOTEBOOK Objectives • To test your genotype at the circadian clock gene Per3 • To learn about the circadian clock, amplify one of your clock genes, and study a possible genetic association with your own sleep phenotype

NOTEBOOK Procedure Overview • Part I – DNA Extraction • Part II – PCR of Period 3 Gene • Part III – Gel Electrophoresis of Period 3 VNTR Region

NOTEBOOK Materials • 2 miniPCR tubes – 1 for DNA Extraction – 1 for PCR Reaction • X-Tract DNA Extraction Buffer • Toothpick • 5X EZ PCR Master Mix ( EZ MM ) • Sleep Lab Primers ( Pri )

NOTEBOOK Morning-Eveningness Score • Calculate and record your morning- eveningness score • Based on your score, what “type” are you? • Does your score match with the sleep onset and wake-time times listed? Explain.

Per3 Gene • Period 3 is a circadian clock gene that has been associated with sleep behavior preferences in humans • Coding region of the gene contains a VNTR (variable number tandem repeat) that is polymorphic (variable) across people • The VNTR region is 54 base pairs in length

VNTR Polymorphisms • Studies have shown that: – 4 copies of the repeat was found at higher frequencies in people with a preference for evening activities – 5 copies of the repeat was found in higher frequencies in people with a preference for morning activities

NOTEBOOK Gel Electrophoresis Procedure and Run • Lane 1 – Load 10 µL of the Ladder • Load 15 µL of PCR product from your PCR sample into a well • Record Lane Number: • Run at 100 volts for ~40 minutes

NOTEBOOK Fragment Size Prediction • What is your predicted genotype for the Per3 gene? • How many bands do you expect to be visible on the gel? • What size is each fragment? Or what size is the fragment? (in base pairs)

NOTEBOOK Electrophoresis Results • Record the results of your electrophoresis experiment: Band number: __________ Fragment size: __________ Genotype: __________

Expected Experimental Results

5X EZ PCR Master mix 5 µL Taq polymerase + PCR Buffer + Mg 2+ + dNTPs Sleep Lab Primers 20 µL Forward and Reverse primers 3 µL DNA Extraction Human Genomic DNA CD 28 µL Add your initials to side of tube