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Labreview.pdf

Edvo-Kit #345

Exploring the Genetics of Taste: SNP Analysis of the PTC Gene Using PCR Experiment Objective:

In this experiment, students will isolate their own DNA and use PCR to amplify a segment of the TAS2R38 gene, which is responsible for detecting the bitter taste of PTC. Digestion of the PCR products and analysis by agarose gel electrophoresis are used to differentiate tasters and non-tasters. Finally, students' genotype is linked to phenotype by tasting the PTC paper.

See page 3 for storage instructions.

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Table of Contents

EDVOTEK, The Biotechnology Education Company, and Dryzyme are registered trademarks of EDVOTEK, Inc. EdvoBead, LyphoPrimer, LyphoTemplate, and UltraSpec-Agarose are trademarks of EDVOTEK, Inc.

Experiment Components 3 Experiment Requirements 4 Implementing the Lab 5

Experiment Procedures Module I Overview 6 Module I: Isolation of DNA from Human Cheek Cells 7 Module II: Amplification of the PTC Region 8 Module III: Restriction Digest of the PTC PCR Product 9 Module IV: Separation of DNA Fragments by Electrophoresis 10 Module V: Determination of Bitter Tasting Ability with PTC Paper 13

Pre-Lab Overview 14 Pre-Lab Preparations 15 Experiment Results and Analysis 21 Answers to Study Questions 22

Appendices 23 A Troubleshooting Guides 24 B Bulk Preparation of Electrophoresis Buffer and Agarose Gels 27 Safety Data Sheets can be found on our website: www.edvotek.com/safety-data-sheets

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Experiment Components

All experiment components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals.

This kit contains enough reagents for 25 DNA

reactions plus 5 controls.

Sample volumes are very small. It is important to quick spin the tube contents in a microcentrifuge to obtain

sufficient volume for pipet- ting. Spin samples for 10-20 seconds at maximum speed.

Perishable Components Storage Check (√)

• PCR EdvoBeads™ Plus* Room Temperature q

A Universal DNA Buffer -20°C Freezer q

B TE Buffer -20°C Freezer q

C LyphoPrimer™ Mix -20°C Freezer q

D LyphoControl™ (Complete PCR control) -20°C Freezer q

E Dryzymes® Restriction Enzyme HaeIII -20°C Freezer q

F Restriction Enzyme Dilution Buffer -20°C Freezer q

G 100 bp ladder -20°C Freezer q

• Proteinase K -20°C Freezer q

• PTC Paper Room temperature q

• Control Taste Paper Room temperature q

NOTE: Components C and D are supplied in our LyphoPrimer™ and LyphoControl™ format. They will require reconstitution prior to use. Be sure to review page 21 in the Instructor's Guide for more details.

* Each PCR EdvoBead™ Plus contains: dNTP Mixture, Taq DNA Polymerase Buffer, Taq DNA Polymerase, MgCl2 , Reaction Buffer

Reagents & Supplies Store all components below at room temperature.

• UltraSpec-Agarose™ q

• TBE Electrophoresis Buffer Powder q

• SYBR® Safe Stain q

• Disposable plastic cups q

• 15 mL Conical Tube q

• Snap-top microcentrifuge tubes q

• Microcentrifuge Tubes (1.5 mL screw-cap tube – use for boiling) q

• 0.2 mL PCR tubes q

• Salt packets q

EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR INSTRUCTOR'S GUIDEEDVO-Kit #345

Requirements (NOT included in this experiment)

• Thermal cycler (EDVOTEK Cat. #540 or #541-542 highly recommended) or three water baths* • Two Water bath for 55°C and 99°C incubations (EDVOTEK® Cat. #539 highly recommended) • Horizontal gel electrophoresis apparatus • D.C. power supply • Balance • Microcentrifuge • UV Transilluminator or Blue Light Visualization System (EDVOTEK® Cat. #557 or #558 highly recommended) • UV safety goggles • Automatic micropipettes (5-50 µL) with tips • Microwave or hot plate • Pipet pump • 250 mL flasks or beakers • Hot gloves • Disposable laboratory gloves • Ice buckets and ice • Distilled or deionized water - 3.7 L (NOTE: for ease of preparation, we recommend purchasing one gallon [3.78 L] of dis-

tilled water.) • Drinking water • Bleach solution

*If you do not have a thermal cycler, PCR experiments can be conducted, with proper care, using three water baths. However, a thermal cycler assures a significantly higher rate of success.

INSTRUCTOR'S GUIDE EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR EDVO-Kit #345

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Implementing the Lab

EXPERIMENT OBJECTIVE

The objective of this experiment is for students to isolate human DNA and use PCR to amplify a segment of the TAS2R38 gene, which is responsible for detecting the bitter taste of PTC.

In MODULE I, Human DNA is collected from student cheek cells after a brief incubation in lysis buffer, followed by boiling the cell suspension. In MODULE II, the human DNA is mixed with PCR primers and a PCR EdvoBead™ PLUS, then PCR is performed in a thermal cycler. Once the TAS2R38 gene has been amplified, the PCR product is mixed with the HaeIII restric- tion enzyme during MODULE III.

Finally, in MODULES IV and V, students will compare their genotype with their phenotype - in this case, the ability to taste PTC. Digested and undigested DNA is analyzed by agarose gel electrophoresis allowing students to detect the presence of a SNP in their TAS2R38 gene. This genotype result is linked to phenotype by tasting the PTC paper.

LAB PREPARATIONS

Prelab preparations begin on page 14. Please review the entire Instructor's Guide before beginning the experiment.

Before running the experiment, confirm that the program on your thermal cycler matches the PCR settings below:

Initial denaturation: 94°C for 4 minutes 94°C for 30 seconds 64°C for 30 seconds 35 cycles 72°C for 30 seconds Final Extension: 72°C for 5 minutes

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This is the INSTRUCTOR'S GUIDE. The Student's Guide does not contain these tips and tricks or prelab preparation instructions.

Instructional

Videos

Learning

Center

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In-Depth Understanding of Module I:

In Module I, the students will be isolating DNA from cheek (Buccal) cells using a quick cell lysis protocol. The cells are collected in a saline solution that has been prepared in advance - it is essential that saline is used here, since other solutions can inhibit the PCR in Module II. Once collected, the cheek cells are suspended in a lysis buffer containing a pH buffer, detergent, and protease. This solution, as well as

two brief incubations, will weaken and then lyse the cells. Finally, a DNA solution is collected by centrifuging the cell lysate. It is important that students collect a relatively pure DNA solution as cell debris from the lysis steps can inhibit PCR. It might be helpful to review the

“Where’s my DNA” flowchart before beginning the experiment to ensure that students do not accidentally discard their DNA.

Resources For additional information about

this experiment, visit: www.edvotek.com/345

For resources on pipetting, isola- tion of cheek cell DNA, and more:

www.edvotek.com/guides- lesson-plans

www.youtube.com/Edvotekinc

Before you start: q Ensure that this lit version

(345.220421) matches the label found in your perishables.

q Review the experiment back- ground with students

q Remind students of safety guidelines and disposal info

for the cheek cell solutions.

Techniques: Before beginning this experiment, students should be proficient in pipetting.

Detailed pre-lab preparation instructions can be found on page 15. Remember to plan for stopping points if you will not be continuing directly to Module II.

IMPORTANT: Only use saline for the student cheek cell rinse. Using sports drinks or other solutions may inhibit the PCR in Module II.

Strategy & Planning

Pre-Lab Preparation

STUDENT PROTOCOL PREVIEW

INSTRUCTOR'S GUIDE EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR EDVO-Kit #345

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Human saliva may transmit disease. We recommend steril- izing all human samples with 10% bleach after completing the experiment.

Remind students to wear gloves and goggles while working in the lab, and to leave the lab area before eating or drinking (after the saline rinse).

Boiling water is hot, use caution!

Safety

Strategy & Planning

Vocabulary Review

Saline - A salt water solution. Resuspend - Place a solid back into solution. Supernatant - The liquid layer above a sedi- ment or solid. Incubate - To maintain a solution at an ideal condition or temperature.

Experimental Assessment

Ask the students to critically analyze each step of the protocol. For example: Why is the saline rinse centrifuged twice, or, why do we only collect 50 µL of the supernatant in step 11?

DNA is very stable when fro- zen. Once the students have completed this module, their samples can be stored in the freezer (-20°C) until needed in Module II.

1) You must use saline for the rinse!

5) After centrifuging you should see at least a faint pellet in each student’s tube. If not, ask the students to swirl their remaining saline solution to resuspend settled cells and repeat the steps 3 and 4 again.

6) Ensure cells are fully resus- pended - no clumps!

9) This does not need to be a rolling boil, but we want the water as hot as possible.

11) Ensure that there is no debris transferred with the DNA supernatant. If you notice any clumps or cloudi- ness in the samples they can be centrifuged again to pellet any debris.

STUDENT PROTOCOL PREVIEW

Stopping Point

EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR INSTRUCTOR'S GUIDEEDVO-Kit #345

Preparing Students for PCR:

PCR is a ubiquitous technique that has become a cornerstone of modern biotechnology. Despite this, many of the details of the PCR process can be obscured behind the relatively simple protocol. Ensure that your students fully understand the science behind PCR by carefully

reviewing the background information or incorporating supplemental lessons. Additional PCR lesson plans can be found at:

www.edvotek.com/site/pdf/PCR_Lesson_Plan.pdf

Picking up where you left off:

q If the samples were frozen at the end of Module I, they can be thawed on ice, or briefly held between the student’s fingers.

q Remind students that these are their extracted DNA samples which will be used for the PCR.

q Review the steps of the prot- col, ensure that each student or pair of students has the necessary reagents.

q Ensure that the thermal cycler is programmed with the correct protocol and is functioning properly.

Detailed pre-lab preparation instructions can be found on page 16. Remember to plan for a stopping point if you will not be continuing directly to Module III.

IMPORTANT: Prepare the controls before beginning the student protocol (see page 16). Controls should be run alongside student samples if possible.

Before You Start

Pre-Lab Preparation

Strategy & Planning

A properly mixed PCR reaction will be a light orange color after step 4. This is a good checkpoint to ensure that students have added the template and primers in the correct volumes.

Once the reaction mixture has been prepared (after step 4) we recommend proceeding through the PCR before stopping the experiment at the end of the module.

PCR is incredibly sensitive - each student must use a fresh pipet tip when mixing their samples.

STUDENT PROTOCOL PREVIEW

INSTRUCTOR'S GUIDE EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR EDVO-Kit #345

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Why Do We Use the HaeIII Restriction Enzyme?

There are hundreds of purified restriction enzymes available for biologists. Many consider- ations go into choosing which enzyme will be used for a specific experiment, including re-

action conditions, the composition of the DNA solution, and, most importantly, the desired cut site in the DNA template. The HaeIII restriction enzyme recognizes the four nucleotide

sequence “GGCC”. For this experiment, HaeIII was chosen for its ability to cut exactly where we would like to analyze a SNP in the TAS2R38 gene. Additional information on the SNP

can be found in the background section provided in the Student's Guide.

Picking up where you left off: q If the samples were frozen

at the end of Module II, they can be thawed on ice or briefly held between the student’s fingers.

q Remember to collect any control samples that were amplified by PCR in Module II, they should also be used in Module III.

Detailed pre-lab preparation instructions can be found on page 18. Remember to plan for a stopping point if you will not be continuing directly to Module IV.

IMPORTANT: The HaeIII restriction enzyme must be rehydrated before per- forming the experiment. Enzyme can be prepared ahead of time and stored until needed. See page 18 for details.

Before You Start

Pre-Lab Preparation

Strategy & Planning

Students will be digesting HALF of their PCR product from Module II. Ensure that students save the undigested portion for analysis in Module IV.

Control samples will be used in this module to confirm that the restriction digest is working as expected. We recommend digest- ing the controls alongside the students samples if possible, but they can be prepared ahead of time and frozen until needed in Module IV.

At the completion of this module, all samples can be stored in the freezer (-20°C) until needed. Ensure that students save BOTH the digested and undigested DNA samples.

STUDENT PROTOCOL PREVIEW

Stopping Point

EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR INSTRUCTOR'S GUIDEEDVO-Kit #345

SYBR® Safe Stain

SYBR Safe is a small molecule dye that serves to enable visualization of nucleic acids. When SYBR Safe binds to DNA, its fluorescence under blue light is greatly enhanced. SYBR Safe should be stored at room temperature protected from light and must be dilut- ed before use. Typical dilutions for SYBR Safe are 1:10,000-1:50,000. In this lab, 1:20,000 is the optimal dilution. Instructions for the 1:20,000 dilution can be found on page 19.

Detailed preparation for agarose gel electrophoresis can be found on page 19. This includes dilution of TBE buffer and SYBR safe.

For bulk preparation of agarose gels, detailed instructions can be found on page 27.

Pre-Lab Preparation

Strategy & Planning

• 7 x 7 cm gels are recommend- ed. Each gel can be shared by 2 students. Place comb in the first set of notches.

• Gels should be carefully monitored during heating to prevent excess evaporation of buffer.

• Agarose should be cooled to 60 degrees before adding diluted SYBR safe. This can be done by swirling the molten agarose in the flask for ~30 seconds.

STUDENT PROTOCOL PREVIEW

SYBR Gels can be stored at 4°C in a sealed plastic bag with 1-2 mL of 1X TBE buffer for up to one week. DO NOT FREEZE the gels as this will destroy them.

Resources

For additional information about this experiment, visit:

www.edvotek.com/345

For resources on electrophoresis:

www.edvotek.com/guides- lesson-plans

www.youtube.com/Edvotekinc

Stopping Point

INSTRUCTOR'S GUIDE EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR EDVO-Kit #345

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Agarose Gels:

While agarose gels might look solid, molecularly they contain many holes and channels that the DNA will migrate through. The higher percentage of gel, the smaller those holes and channels become. High percentage gels, like the 2% one used in this ex- periment, are able to more accurately separate small fragments of DNA than lower (0.8-1%) gels.

Picking up where you left off: q If the samples were frozen

after Module III, they can be thawed at room temperature or briefly held between the student's fingers.

q Remember to thaw the con- trol samples alongside the student samples

q If the gels were pre-cast, they will need to be re-attached to the gel casting tray. This can be done by melting any extra agarose, or a pack of jello. Place a few drops of molten agarose or jello onto the gel casting tray and the pre-cast gel on top.

Before You Start

Strategy & Planning

Students will load both their digested and undigested samples. Control DNA, digested and/or undigested, should be loaded into every gel.

STUDENT PROTOCOL PREVIEW

View our playlist of how-to electrophoresis videos on

YouTube:

www.youtube.com/Edvotekinc

If not visualizing the gel immedi- ately, store for up to 1 week at 4°C in a sealed plastic bag with 1-2 mL buffer. For more info see:

https://blog.edvotek. com/2019/03/29/edvotek/

Stopping Point

EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR INSTRUCTOR'S GUIDEEDVO-Kit #345

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Module IV: Separation of DNA Fragments by Electrophoresis, continued

VISUALIZING THE SYBR® GEL

13. SLIDE the gel off the casting tray onto the viewing surface of the transilluminator and turn the unit on. ADJUST the brightness to the desired level to maximize band visualization. DNA should appear as bright green bands on a dark background.

14. PHOTOGRAPH the results. 15. REMOVE and DISPOSE of the gel and CLEAN the transilluminator surfaces with

distilled water.

dH2O

Be sure to wear UV goggles if using a

UV transilluminator.

INSTRUCTOR'S GUIDE EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR EDVO-Kit #345

Module V: Determination of Bitter Tasting Ability with PTC Paper

In this final Module you will taste strips of control and PTC paper to determine your phenotype. First, you will taste a con- trol strip of paper – it is important to taste the control before the PTC-coated strip since the paper will have a slight taste. Next, you will taste the PTC paper, paying attention to any differences between it and the control paper.

PROCEDURE:

1. TASTE the Control strip of paper first. RECORD your thoughts on the taste.

2. TASTE the PTC strip of paper. RECORD your thoughts on the taste.

3. COMPARE the taste of the Control and the PTC paper.

• Notice what the PTC paper tastes like compared to the Control paper: intensely bitter, somewhat bitter, or taste- less.

• If you are a taster, the PTC paper strip will be bitter. Non-tasters will not notice a difference between the strips of paper.

ANALYZE THE RESULTS:

1. Compare your genotype, from Module IV, to the phenotype observed when you taste the PTC strips. Do your results match?

2. Are you a homozygous bitter taster, a heterozygous bitter taster, or a non-taster?

EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR INSTRUCTOR'S GUIDEEDVO-Kit #345

Pre-Lab Overview

INSTRUCTOR'S GUIDE EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR EDVO-Kit #345

This experiment will require 3-4 class periods to complete the student experiment. Many reagents can be prepared in advance, but others require special considerations. Please consult the table below and the detailed instructions in the Instructor's Guide for more information.

Module I: Isolation of DNA from Cheek Cells

Prepare and aliquot Lysis Buffer.

Prepare on the day the students will be performing the experiment OR freeze for up to one week.

15 min.

5 min.

Module IV: Separation of DNA Fragments by Electrophoresis

Module V: Determination of Bitter Tasting Ability

Prepare TBE buffer.

45 min.

Module II: Amplification of the PTC Region

Prepare and aliquot various reagents (Primer, PCR Edvo- Beads™, etc.).

Prepare and aliquot saline and plastic tubes

Anytime before performing the experiment. 30 min.

Up to one week before performing the experiment. Diluted primers should be frozen until needed.

Up to one week before performing the experiment.

Distribute PTC taste strips and Control taste strips.

5 min.During the class period you perform the experiment.

30 min.

15 min.

Equilibrate water baths at 55° C and boiling.

Program Thermal Cycler.

Anytime before performing the experiment.

Module III: Restriction Digest of the PTC PCR Product

Prepare and aliquot HaeIII Restriction enzyme.

Prepare on the day the students will be performing the experiment OR freeze for up to one week.

15 min.

15 min.Equilibrate water bath at 37° C.

Anytime before performing the experiment.

Prepare molten agarose and pour gels (OPTIONAL) or aliquot agarose, TBE, and SYBR® Safe.

Aliquot 100 bp ladder.

Red = Prepare immediately before module. Yellow = Prepare shortly before module. Green = Flexible / prepare up to a week before the module.

Pre-Lab Preparations: Module I In Module I, the students will isolate DNA from their cheek cells. The easiest, and recommended, method to collect cheek cells involves a brief rinse with a saline solution. The saline rinse will be centrifuged briefly to isolate the cheek cells, which are then mixed with a lysis buffer and incubated at 55˚C and 99˚C. Finally, the students will centrifuge the cell lysate and collect the DNA-containing supernatant. The isolated DNA can be safely stored at -20˚C (freezer) until needed for PCR in Module II.

Before beginning Module I the instructor must prepare the Saline Solution and Lysis Buffer. See below for preparation instructions for both solutions.

Preparation of Saline Solution

NOTE: Saline solution MUST be used for cheek cell wash. Sports drinks will inhibit amplification of DNA by Polymerase Chain Reaction in Module II. If you have used sports drinks for the cheek cell wash, please DISCARD the samples and REPEAT the DNA extraction with saline solution

1. To prepare the saline solution, dissolve all eight salt packets (~4 g) in 500 mL of drinking water. Cap and invert bottle to mix.

2. Aliquot 10 mL of saline solution per cup. Distribute one cup per student.

Preparation of Lysis Buffer

NOTE: The Lysis Buffer must be mixed with Proteinase K before performing the experiment. Once prepared, the Lysis should be used the same day or frozen.

1. Add 100 µL of Universal DNA buffer (A) to the tube of Proteinase K and allow the sample to hydrate for several minutes. After the sample is hydrated, pipet up and down several times to thoroughly mix the material.

2. Transfer the entire amount of the rehydrated Proteinase K solution to a 15 mL conical tube containing an additional 4 mL of Universal DNA buffer (A).

3. Invert the tube several times to mix. Label this tube “Lysis Buffer”.

NOTE: The Lysis Buffer should be red and free of any undissolved clumps.

4. Aliquot 300 µL of Lysis Buffer into 13 labeled microcentrifuge tubes to be shared by pairs of students.

NOTE: At this point, the Lysis Buffer should be stored on ice for use within the same day (up to 6 hours) or frozen.

5. Distribute one tube of “Lysis Buffer” to each student pair. If frozen, the Lysis Buffer can be quickly thawed in a 37°C water bath or by students warming the tube in their hands.

DISINFECTING LABORATORY MATERIALS: Contaminated laboratory waste (saliva solution, cup, pipet, etc.) must be disinfected with 15% bleach solution prior to disposal. Be sure to properly dispose any biological samples according to your institutional guidelines.

FOR MODULE I Each student should receive: • One cup containing 10 mL of

saline solution • One screw-cap tube • One microcentrifuge tube

Reagents to be shared by two students: • 300 µL Lysis buffer • 15% bleach solution

Warning !! Remind students to only use screw-cap tubes when boiling their DNA samples. The snap- top tubes can potentially pop open and cause injury.

NOTE: Accurate pipetting is critical for maximizing successful experiment results. This ex- periment is designed for stu- dents who have had previous experience with micropipet- ting techniques and agarose gel electrophoresis.

If students are unfamiliar with using micropipets, we recom- mended performing Cat. #S-44, Micropipetting Basics or Cat. #S-43, DNA DuraGel™ prior to conducting this ad- vanced level experiment.

EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR INSTRUCTOR'S GUIDEEDVO-Kit #345

Pre-Lab Preparations: Module II In Module II, the students will perform PCR on the DNA that they isolated in Module I. The PCR mixture includes the student DNA, a primer mixture specific to a 221 bp region of the TAS2R38 gene, and a PCR EdvoBead™ PLUS.

The PCR primers are provided as a lyophilized mixture that must be rehydrated by the instructor before performing the experiment. The PCR EdvoBeads™ PLUS can be dis- tributed prior to setting up the PCR – students or instructors can gently transfer the PCR EdvoBeads™ PLUS using gloved hands. Alternatively, beads can be gently "poured" from the vial into individual PCR tubes. After distributing the beads, it is important to close the PCR tubes securely to prevent the beads from absorbing moisture and becoming hard to resuspend prior to the experiment.

NOTE: The PCR EdvoBeads™ PLUS are fragile, use care to not crush the beads while transferring to a PCR tube.

This experiment contains LyphoControl™ Complete PCR control samples that should be prepared and run alongside the student PCR samples. LyphoControl™ samples are premixed with all PCR components and are ready to run immediately after rehydration. We highly recommend running the controls alongside student samples if possible, although they can also be run before or after the student PCR.

Preparation of the PTC Primer Mix

1. Thaw the TE Buffer (B) and mix well. 2. Ensure that the lyophilized solid is at the bottom of the LyphoPrimer™ tube (C). If not, centrifuge the tube at full speed

for 10 seconds. 3. Add 1.2 mL of TE Buffer (B) to the tube. Cap and mix well, then place on ice. The solution should be light orange and

clear of any solid pieces. 4. Label 13 microcentrifuge tubes “PTC Primer”. Aliquot 90 µL of the diluted Primer Mix into the 13 microcentrifuge tubes.

Place the tubes on ice until they are needed. If you would like to prepare the primers ahead of time, they can be ali- quoted and then frozen until needed.

5. Distribute one tube of diluted PTC Primer to each student pair.

Preparation of the LyphoControl™

NOTE: This kit includes enough reagents to set up 6 control reactions. At least one control reaction should be performed per class to confirm that PCR was successful.

1. Ensure that the lyophilized solid is at the bottom of the LyphoControl™ tube (D). If not, centrifuge the tube at full speed for 10 seconds.

2. Add 320 µL of TE Buffer (B) to the tube. Cap and mix well then place on ice. The solution should be light red and clear of any solid pieces.

3. Dispense 50 µL of the diluted control mixture for each control reaction.

NOTES: • The LyphoControl™ already contains all necessary PCR components and does not need a PCR EdvoBead™ PLUS. Once

diluted, the LyphoControl™ is ready to be amplified by the PCR alongside student PCR samples, if there is room, or run prior to the student experiment. After PCR, the control reactions can be stored at -20°C until needed in Module III.

• Distilled water can be used to rehydrate the lyophilized components. Just be sure that the proper volumes are used.

FOR MODULE II Each student should receive: • One PCR tube and PCR EdvoBead™ PLUS

Reagents to be shared by two students: • 90 µL PTC Primer mix

INSTRUCTOR'S GUIDE EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR EDVO-Kit #345

Programming the Thermal Cycler

The Thermal cycler should be programmed as outlined in Module II in the Student’s Experimental Procedure.

• Accurate temperatures and cycle times are critical. A pre-run for one cycle (takes approximately 3 to 5 minutes) is rec- ommended to check that the thermal cycler is properly programmed.

• For thermal cyclers that do not have a heated lid, it is necessary to place a layer of oil or wax above the PCR reactions in the microcentrifuge tubes to prevent evaporation. Visit our website for more information.

NOTE: The PCR cycling conditions may have changed. Before running the experiment, confirm that the program matches the settings below:

• Initial denaturation 94°C for 4 minutes • 94°C for 30 seconds • 65°C for 30 seconds 35 cycles • 72°C for 30 seconds • Final Extension 72°C for 5 minutes

Pre-Lab Preparations: Module II, continued

EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR INSTRUCTOR'S GUIDEEDVO-Kit #345

Pre-Lab Preparations: Module III RESTRICTION DIGEST OF THE PTC PRODUCT

During Module III students will digest some of their PCR product, saved from Module II, using the HaeIII restriction enzyme. For best results, the HaeIII Dryzyme® restriction enzyme must be prepared shortly before use and kept on ice, or stored at -20°C immediately after aliquoting. Each student will receive a single tube of the rehydrated enzyme for use during the experiment.

Remind students to save the remaining, undigested sample for use in Module IV. Once the digest has completed the di- gested and undigested samples can be stored together at -20°C (freezer) until needed.

Dilution of HaeIII Restriction Enzyme

1. Make sure that the solid material is at the bottom of the Dryzyme® Restriction En- zyme HaeIII tube (E). If not, centrifuge the tube at full speed for 20 seconds.

2. Add 200 µL of Restriction Enzyme Dilution Buffer (F) to the tube containing the HaeIII Dryzyme®.

3. Allow the sample to hydrate for 1 minute.

4. Mix the tube for 30 seconds (gently vortex or tap bottom of the tube) and set on ice for 1 minute.

5. Centrifuge the rehydrated enzyme for 20 seconds at max. speed.

6. Dispense 5 µL of the HaeIII Restriction Enzyme into 25 tubes. Label these tubes “HaeIII”

NOTE: The rehydrated restriction enzyme can be stored on ice or in the refrigerator for up to 6 hours, or frozen for up to one week. We do not recommend overnight storage in the refrigerator.

FOR MODULE III Reagents to be shared by two students: • Two tubes of HaeIII Restriction Enzyme

INSTRUCTOR'S GUIDE EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR EDVO-Kit #345

Pre-Lab Preparations: Module IV SEPARATION OF DIGESTION PRODUCTS BY ELECTROPHORESIS

Preparation of Electrophoresis Buffer:

For this experiment, we recommend preparing the TBE electrophoresis buffer in bulk for sharing by the class. Unused diluted buffer can be used at a later time.

1. Measure 3.7 L of distilled or deionized water and place in a large vessel. (NOTE: If using purchased water in a gallon jug, remove and discard 80 mL water.)

2. Add the entire amount of TBE electrophoresis buffer powder to the vessel and mix well. 3. Label the vessel as "1X Electrophoresis Buffer (TBE)". 4. Use within 60 days of preparation.

SYBR® Safe Stain Preparation: Prepare diluted SYBR® Safe by adding 400 µL of 1X TBE buffer to the tube of SYBR® Safe and tapping the tube several times to mix. Diluted SYBR® Safe will be used during agarose gel preparation.

Preparation of Agarose Gels:

This experiment requires one 2.0% agarose gel for every group of two students (13 gels total). 7 x 7 cm gels are recom- mended. You can choose whether to prepare the gels in advance or have the students prepare their own. Allow 30-40 minutes for this procedure.

Individual Gel Preparation: Each student group can be responsible for casting its own individual gel prior to conducting the experiment (see Module IV in the Student’s Experimental Procedure). Each 7 x 7 cm gel will require 25 mL of 1X TBE buffer, 0.5 g of agarose powder, and 25 µL of diluted SYBR® Safe Stain.

Batch Gel Preparation: To save time, a larger quantity of agarose solution can be prepared for sharing by the class. See Appendix B for instructions.

Preparing Gels in Advance: Gels may be prepared ahead and stored for later use. Solidified gels can be stored for up to 1 week in the refrigerator in water-tight bags with a small amount of buffer to prevent drying. We recommend adding 2 mL of buffer to the bag; excess buffer can lead to diffusion of SYBR® Safe out of the gels.

Do not store gels at -20º C as freezing will destroy them.

Gels that have been removed from their trays for storage should be “anchored” back to the tray with a few drops of molten agarose before being placed into the tray. This will prevent the gels from sliding around in the trays and the chambers.

FOR MODULE IV Each group receives: • 1X TBE buffer • UltraSpec-Agarose™

Powder • Tube of diluted SYBR® Safe (25 µL) • 100 bp ladder (30 µL)

EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR INSTRUCTOR'S GUIDEEDVO-Kit #345

Pre-Lab Preparations: Module IV, continued

Additional Materials:

• Aliquot 30 µL of the 100 bp ladder (G) into labeled microcentrifuge tubes and distribute one tube of 100 bp ladder per gel.

Each 2.0% gel should be loaded with the 100 bp ladder and samples from two students. The control PCR reaction can also be loaded in one of the wells. If desired, you can designate one student or pair of students from each gel to load the con- trols.

Photodocumentation of DNA (Optional):

Once gels are stained, you may wish to photograph your results. There are many different photodocumentation systems available, including digital systems that are interfaced directly with computers. Specific instructions will vary depending upon the type of photodocumentation system you are using.

INSTRUCTOR'S GUIDE EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR EDVO-Kit #345

Experiment Results and Analysis

Lane Sample

Bands range in size from 100 bp – 4000 bp in 100 bp increments. High intensity reference band at 500 bp.

221 bp

177, 44 bp

221 bp

177, 44 bp

100 bp ladder

Control DNA, uncut

Control DNA, cut

Student DNA, uncut

Student DNA, cut

Size

Lane Sample

Bands range in size from 100 bp – 4000 bp in 100 bp increments. High intensity reference band at 500 bp.

221 bp

221, 177, 44 bp

221 bp

100 bp ladder

Student 1 DNA, uncut

Student 1 DNA, cut

Student 2 DNA, uncut

Student 2 DNA, cut

Size

• Homozygous Taster: Both copies of the gene contained the polymorphism, allowing it to be digested by HaeIII. • Heterozygous Taster: One copy of the gene contained the polymorphism, allowing it to be digested by HaeIII. The other copy of the

gene did not have the restriction site and was not digested • Homozygous Non-taster: Neither copy of the gene contained the polymorphism, so HaeIII could not digest this DNA. NOTE: The 221 and 177 bp bands will appear brighter than the 44 bp band.

221 bp Fragment 177 bp Fragment

44 bp Fragment

Hae III Restriction DigestHae III Restriction Digest

Agarose Gel Electrophoresis

PCR Amplification of PTC region

Extraction of DNA from Buccal cells

PTC TASTER: Homozygous Taster (TT) = sizes of 177 & 44 bp Heterozygous Taster (Tt) = 1 allele remains uncut at 221bp while the other allele cuts and generates fragments of 177 bp and 44 bp.

PTC NON-TASTER: Homozygous recessive (tt) = remains uncut at 221 bp

GGCGGGCACT

GGCGGCCACT

Non-Taster (tt)Homozygous Taster (TT)

GGCGG CCACT

PCR Product 221 bpPCR Product 221 bp

221 bp Fragment (remains uncut)

44 bp Fragment 177 bp Fragment

Hae III Restriction Digest

Agarose Gel Electrophoresis

PCR Amplification of PTC region

Extraction of DNA from Buccal cells

GGCGGCCACT

Heterozygous Taster (Tt)

PCR Product 221 bp

GGCGGGCACT +

PCR Product 221 bp

PCR Amplification of PTC region

Extraction of DNA from Buccal cells

Agarose Gel Electrophoresis

GGCGG CCACT

44 bp Fragment 177 bp Fragment 221 bp Fragment

Lane 1 (TT)

Lane 2 (Tt)

Lane 3 (tt)

NOTE: In some samples, a diffuse, low molecular weight band known as a "primer dimer" may be present. This is a PCR artifact and can be ignored.

EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR INSTRUCTOR'S GUIDEEDVO-Kit #345

Study Questions & Answers

1. How is PCR used to determine human genetics and identify polymorphisms in DNA?

By using PCR scientists can amplify a specific region of the DNA and test that DNA fragment for restriction enzyme digestion. Polymorphisms can also be detected when primer pairs are designed to target a specific region that varies by size.

2. What are the three steps in a PCR cycle and what does each step accomplish?

The three steps are: denaturation, annealing, and extension. These three steps constitute one PCR cycle. Each cycle doubles the amount of the target DNA. Denaturation disrupts hydrogen bonds between DNA strands, separating the DNA strands. Annealing allows the primers to bind to the DNA template. Finally, extension allows the Taq polymerase to synthesize new DNA.

3. Based on what you have learned about the genotype of TAS2R38 and its phenotype, fill in the table below:

4. Based on your results, what is your genotype? Why? What is your phenotype? Why? How about your lab part- ners?

Answer will vary.

5. Do the control and PTC paper tasting results correlate with the DNA digest findings in your ability to taste? How about your lab partner?

Answer will vary. However, the ability to taste PTC bitterness should correlate with the genotype.

6. Enter your classroom data in the Table shown below:

Phenotype Genotype Strong Taster Weak taster Non-taster TT (homozygous) Tt (heterozygous) tt (homozygous)

Answers will depend on your classroom data.

7. Considering that not everyone who can taste PTC tastes it the same way, what does this tell you about classi- cal dominant/recessive inheritance?

The PTC taster phenotype is not absolute. For example, the taste threshold for homozygous and heterozygous individ- uals often varies. In addition, some individuals seem to experience a delayed taste response to PTC. These phenotypes might indicate additional factors that are influencing taste perception.

Genotype Phenotype # of DNA bands predicted Size of bands (bp) TT Homozygous Taster 2 bands 177 bp & 44 bp

Tt Heterozygous Taster 3 bands 221 bp, 177 bp, & 44 bp

tt Homozygous Recessive 1 band 221 bp Non-taster

INSTRUCTOR'S GUIDE EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR EDVO-Kit #345

EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR APPENDICESEDVO-Kit #345

A EDVOTEK® Troubleshooting Guide B Bulk Preparation of Electrophoresis Buffer and Agarose Gels

Safety Data Sheets can be found on our website: www.edvotek.com/safety-data-sheets

Appendices

https://www.edvotek.com/safety-data-sheets

mailto:info%40edvotek.com?subject=

https://www.edvotek.com

http://www.edvotek.com

APPENDICES EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR EDVO-Kit #345

Appendix A EDVOTEK® Troubleshooting Guides

DNA EXTRACTION

PROBLEM: CAUSE: ANSWER:

There is no cell pellet after centrifuging the cheek cell suspension.

Not enough cheek cells in suspension Mouth must be vigorously rinsed for at least 60 sec. to harvest loose cheek cells.

Sample not centrifuged fast enough Spin cells at maximum speed for 2 min. If your centrifuge’s max RCF is less than 2000 x g, spin at highest available speed for 4 min.

The extracted DNA is very cloudy.

Cellular debris from pellet transferred to tube

Centrifuge sample again and move supernatant to a fresh tube. Take care to avoid pellet.

Cellular debris not separated from supernatant

Centrifuge sample again. If possible, centrifuge at a higher speed. Move cleared supernatant to a fresh tube.

Poor DNA extraction

Samples not mixed well enough during extraction

In addition to flicking the tube, vortex or pipet up and down to mix the sample.

Proteinase K inactive because it was prepared too far in advance.

Prepare Lysis buffer the same day that it will be used or immediately freeze until needed.

Water baths not at proper temperature Use a thermometer to confirm water bath set point.

Not enough DNA Ensure students are centrifuging saline rinse TWICE during Module I.

Sports drink was used for DNA extraction.

Repeat DNA extraction with saline solution.

EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR APPENDICESEDVO-Kit #345

Appendix A EDVOTEK® Troubleshooting Guides

RESTRICTION ENZYME DIGESTION

PROBLEM: CAUSE: ANSWER:

Undigested or incompletely digested DNA

Impure DNA – some contaminants (EDTA, salts) might partially or completely inhibit activity of HaeIII restriction enzyme

Poor DNA extraction. Extract new DNA. Cheek cell extraction usually results in higher DNA yield.

Smearing of digested DNA on gel

Nuclease contamination Care should be taken to avoid cross contamination when setting up reactions.

Agarose running conditions Use fresh electrophoresis buffer and appropriate voltage.

Unexpected cleavage pattern

Improper dilution of enzyme Ensure that HaeIII restriction enzyme was correctly diluted.

Improper addition of enzyme Ensure that correct amount of HaeIII restriction enzyme was added to the restriction digest.

Incorrect incubation temperature Use a thermometer to confirm water bath temperature and adjust, if necessary.

DNA sample is contaminated Prepare a new DNA sample.

Abbreviated incubation time Samples must be incubated 30 min. Slower incubations may lead to incomplete digestions.

APPENDICES EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR EDVO-Kit #345

PCR AND ELECTROPHORESIS

PROBLEM: CAUSE: ANSWER:

There is very little liquid left in tube after PCR.

Sample has evaporated.

Make sure the heated lid reaches the appropriate temperature.

If your thermal cycler does not have a heated lid, overlay the PCR reaction with oil or wax.

Make sure students close the lid of the PCR tube properly.

After staining, the ladder and control PCR products are visible on the gel but some student samples are not present.

Some student samples have more/less amplification than others.

Student DNA sample was not concentrated enough.

Poor DNA extraction. Repeat Module I (Isolation of DNA from Human Cheek Cells).

Pipetting error. Make sure students pipet 40 µL primer mix and 10 µL extracted DNA into the 0.2 mL tube.

The ladder, control DNA, and student PCR products are not visible on the gel.

The gel was not prepared properly.

The gel was not stained properly.

Ensure that the electrophoresis buffer was correctly diluted.

Gels of higher concentration (> 0.8%) require special attention when melting the agarose. Make sure that the solution is completely clear of “clumps” and glassy granules before pouring gels.

The proper buffer was not used for gel preparation. Make sure to use 1x Electrophoresis Buffer.

Ensure that SYBR® Safe was added to the gel before casting. If staining with FlashBlue™, either re-stain with FlashBlue™ solution or destain until bands are seen.

Wrong volumes of DNA and primer added to PCR reaction. Practice using micropipets.

Contact the manufacturer of the electrophoresis unit or power source.

Student DNA sample was degraded. If DNA is not used right after extraction, store sample at -20° C.

Concentration of DNA varies by sample. There is an inherent variability in the extraction process.

Low molecular weight band in PCR samples. Primer dimer Low concentration of extracted DNA in PCR reaction.

DNA bands were not resolved.

To ensure adequate separation, make sure the tracking dye migrates at least 4 cm on 7 x 7 cm gels and 6 cm on 7 x 14 cm gels.

Be sure to run the gel the appropriate distance before staining and visualizing the DNA.

Malfunctioning electrophoresis unit or power source.

Sports drink was used for DNA extraction.

Repeat DNA extraction with saline solution.

Appendix A EDVOTEK® Troubleshooting Guides

EXPLORING THE GENETICS OF TASTE: SNP ANALYSIS OF THE PTC GENE USING PCR APPENDICESEDVO-Kit #345

BULK 1X TBE ELECTROPHORESIS BUFFER

For this experiment, we recommend preparing the 1X TBE Electrophoresis Buffer in bulk for sharing by the class. Unused diluted buffer can be used at a later time.

1. Measure 3.7 L of distilled or deionized water and place in a large vessel. (NOTE: If using purchased water in a gallon jug, re- move and discard 80 mL water.)

2. Add the entire amount of TBE Electrophoresis Buffer powder to the vessel and mix well. 3. Label the vessel as "1X TBE Electrophoresis Buffer". 4. Use within 60 days of preparation.

BATCH AGAROSE GELS (2.0%)

Bulk preparation of 2.0% agarose gel is outlined in Table D.

1. Measure 400 mL of 1X TBE Electrophoresis Buffer and pour into a 500 mL flask. 2. Pour 8.0 g of UltraSpec-Agarose™ into the prepared buffer. Swirl to disperse clumps. 3. With a marking pen, indicate the level of solution volume on the outside of the flask. 4. Heat the agarose solution as outlined previously for individual gel preparation. The heating

time will require adjustment due to the larger total volume of gel buffer solution. 5. Cool the agarose solution to 60°C with swirling to promote even dissipation of

heat. If evaporation has occurred, add distilled water to bring the solution up to the original volume as marked on the flask in step 3.

6. If staining with SYBR® Safe, add the entire volume of diluted SYBR® Safe from page 19 to the cooled agarose and mix well.

7. Dispense the required volume of cooled agarose solution for casting each gel. Measure 25 mL for each 7 x 7 cm gel, 50 mL for each 7 x 14 cm gel. For this experiment, 7 x 7 cm gels are recommended.

8. Allow the gel to completely solidify. It will become firm and cool to the touch after approxi- mately 20 minutes. Then proceed with preparing the gel for electrophoresis. Alternatively, gels can be stored in water-tight plastic bags with 2 mL of 1X electrophoresis buffer for up to 1 week in the refrigerator.

Appendix B Bulk Preparation of Electrophoresis Buffer and Agarose Gels

Batch Prep of 2.0% UltraSpec-Agarose™

8.0 g 400 mL

60˚C NOTE: The UltraSpec-Agarose™ kit component is usually labeled with the amount it contains. Please read the label carefully. If the amount of agarose is not specified or if the bottle's plastic seal has been broken, weigh the agarose to ensure you are using the correct amount.

To save time, the electrophoresis buffer and agarose gel solution can be prepared in larger quantities that the whole class can share. Leftover diluted buffer can be used at a later time and solidified agarose gel solution can be remelted.