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

Lab 11: Morning Lark or Night Owl?

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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
• rganisms have circadian clocks to help

transition between light and darkness

Objectives

• To test your genotype at the circadian clock

gene Per3

• To learn about the circadian clock, amplify one
• f your clock genes, and study a possible

genetic association with your own sleep phenotype

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Procedure Overview

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

Region

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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)

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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.

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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

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

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Fragment Size Prediction

• What is your predicted genotype for the Per3

gene?

• How many bands do you expect to be visible
• n the gel?
• What size is each fragment? Or what size is

the fragment? (in base pairs)

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Electrophoresis Results

• Record the results of your electrophoresis

experiment: Band number: __________ Fragment size: __________ Genotype: __________

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Expected Experimental Results

5 µL 20 µL 3 µL

CD

5X EZ PCR Master mix

Taq polymerase + PCR Buffer + Mg2+ + dNTPs

Sleep Lab Primers

Forward and Reverse primers Add your initials to side of tube

DNA Extraction

Human Genomic DNA

28 µL