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EVT 2011_06_07AM
EDVO-Kit #
370
Real Time PCR
Storage: See Page 3 for specific storage instructions
Experiment Objective:
The objective of this experiment is to afford students theopportunity to understand the process of Real Time PCR
and to learn the basic principles of DNA amplificationby the Polymerase Chain Reaction.
The Biotechnology Education Company ®
EDVOTEK, Inc. • 1-800-EDVOTEK • www.edvotek.com
Real Time PCR
EVT 2011_06_07AM
2
370370EDVO-Kit #
EDVOTEK - The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com
Table of Contents
Page
Experiment Components 3Experiment Requirements 3Background Information 4 Experiment Procedures Experiment Overview and General Instructions 8
Experimental Procedures 10 Study Questions 12 Instructor's Guidelines 13 Pre-Lab Preparations 14 Experiment Results and Analysis 15
Study Questions and Answers 16
Appendices PCR Experimental Success Guidelines 17
PCR Using Three Waterbaths 19 Preparation and Handing of PCR Samples with Wax 20 Material Safety Data Sheets 21
All 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 EXPERIMENT DOES NOT CONTAIN HUMAN DNA. None of the experiment components are derived from human sources.
EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of EDVOTEK, Inc.. Ready-to-Load and UltraSpec-Agarose are trademarks of EDVOTEK, Inc.
Real Time PCR
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FAX: (301) 340-0582 • email: info@edvotek.com
370EDVO-Kit #
Experiment Components
Contents: Storage:
A Tubes with PCR reaction pellets™ Room Temp. Each PCR reaction pellet™ contains: • dNTPMixture • Taq DNA Polymerase Buffer • Taq DNA Polymerase • MgCl2B Primer mixture freezer C DNA Standard [1 µg/µl] freezer D DNA Template freezer E UltraPure Water freezer F TE Buffer freezer
Dilute Ethidium Bromide Room Temp.Spot Plates for DNA samples Room Temp. DNA Binding solution Room Temp.Microtest Tubes
Requirements:
• Thermalcycler(EDVOTEK Cat. # 541 highly recommended)• Pipettorsandtips• Hand-heldUVilluminator• Disposablegloves• UV-protectivegoggles• Pipettes• Glassflaskforheatingagarose• Heat-insulatedmittorbottleholder• Microwaveoven
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2006, 2007, 2010, 2011 EDVOTEK, Inc., all rights reserved EVT 2011_06_07AM
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370Real Time PCR
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Real Time PCR
The polymerase chain reaction (PCR) is a universally used method for ampli-fying distinct DNA sequences. Those sequences can be actual genes or spe-cific DNA markers, including DNA repeats, that can be used for identification of individual persons or as tags for genetic diseases. Since its invention in 1986, PCR has revolutionized all subspecialties of the life sciences, including medicine, forensics, and biology, in addition to becoming an invaluable tool for basic scientific research.
The basis for PCR is a heat-stable DNA polymerase (usually Taq polymerase) that can withstand high temperatures. The underlying reaction is shown in Figure 1. The first step involves denaturation of the DNA sample to be test-ed (e.g., viral DNA present in a patient blood sample). Denaturation (Figure 1, double arrow) involves separation of the two DNA strands at near boiling temperatures, generally around 95°C, at which the Taq polymerase remains stable. The temperature is then lowered to allow binding (annealing) of the sequence-specific primers (Figure 1) to the source DNA (“template”). The primers are designed to be complementary only to the sequence to be am-plified, called the target. An example of a target sequence would be a gene that is unique to a specific virus. If the target is not present in the sample (e.g., a specific viral gene is absent), the primers will not bind and the reac-tion will fail to proceed. Following primer binding during the second PCR (annealing) step, the temperature is then raised to approximately 72°C. At this temperature, the Taq polymerase can add new nucleotides (bases at-tached to sugar molecules) to the primers, in a process known as extension. This extension step creates new DNA strands between each annealed primer (Figure 1). The entire process is then repeated a number of times (“cycles”), resulting in a doubling of the number of target molecules following each round of denaturation, annealing, and extension (Figure 1, illustrating cycles 1-3). The entire procedure is generally performed for 30 - 40 cycles, resulting in an ideal amplification of 230 - 240 times the original DNA; in reality, how-ever, the actual amplification is lower, due to imperfect reaction conditions.
In addition to merely amplifying a specific DNA sequence, it is often desir-able to determine the actual amount of that sequence that is present in a specimen prior to amplification (a process called quantitation). For example, physicians often wish to establish the exact level, or titer, of a particular bacteria or virus present in a specific patient sample. Another instance is the need of researchers to determine the extent that a specific gene is “turned on,” i.e., how much RNA is being made from that particular gene. To allow for such a determination, the RNA is copied (in a test tube) into complementary DNA (cDNA) using a process known as reverse transcription. Subsequently, the cDNA is then amplified by PCR to (indirectly) assess how much of the RNA was originally present. This process is known as reverse transcription PCR, or RT-PCR, and can be used to ascertain the amount of ex-pression of a specific gene. By quantitating how much cDNA (and thus how much RNA) of a specific gene is present in a cell, one can assess the extent of expression of that gene.
5Real Time PCR
370EDVO-Kit #
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Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2006, 2007, 2010, 2011 EDVOTEK, Inc., all rights reserved EVT 2011_06_07AM
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Real Time PCR
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2006, 2007, 2010, 2011 EDVOTEK, Inc., all rights reserved EVT 2011_06_07AM
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370Real Time PCR
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Real Time PCR
To quantitate DNA or cDNA, investigators now primarily use a method known as quantitative PCR, or qPCR. This technique is also often referred to asrealtimePCRandusesaninstrumentthatcouplesPCRwithfluorescencedetection. In real time PCR, the amount of PCR product is detected during theactualprocessofamplification(i.e.,inrealtime),usingafluorescentdyeknownasSYBRgreen,whichfluoresces(glows)whenitbindstothedouble-stranded PCR product (Figure 2A). At a specific PCR cycle number, called the thresholdcycle,theSYBRgreenfluorescencewillbecomedetectable(Figure2B, the point at which trace rises sharply above background). The threshold cycle directly depends on how much template DNA (or cDNA) was originally present. Thus, the lower the threshold cycle, the more DNA was initially present in the sample. Instruments used for qPCR are quite complex, using a lasertoirradiatetheSYBRgreenandafluorescencesensor(calledadetec-tor),connectedtoacomputer,tomonitortheSYBRgreenfluorescence.Asa result, such instruments generally cost tens of thousands of dollars and require special training for use and data analysis. One can, however, simu-late qPCR by using other less expensive DNA dyes (such as ethidium bromide) to monitor the formation of a PCR product. In such a simulation, samples can be removed from the PCR reaction and “spotted” on a solid surface. By comparing the spots to standards (known amounts of DNA and also stained with ethidium bromide), one can estimate the amount of DNA in the PCR product.
Figure 2: Quantitative PCRA) SYBR Green dye (stars) is nonfluorescent in solution. However, in the presence of double stranded DNA, fluorescence increases. B) At a specific PCR cycle, called the threshold cycle, fluorescence will become detectable above background.
7Real Time PCR
370EDVO-Kit #
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Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2006, 2007, 2010, 2011 EDVOTEK, Inc., all rights reserved EVT 2011_06_07AM
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Real Time PCR
In this experiment, students will determine the amount of DNA in simulated patient samples, to determine levels (“titers”) of a viral infection. Here, small amounts of the PCR reaction will be removed after 0, 10, 20, 30, & 40 PCR cycles. Those reaction portions will be “spotted” on a Spot Plate and compared to standards containing known amounts of DNA. Rather than SYBR Green, DNA will be stained with ethidium bromide, a dye commonly used for detecting DNA in agarose gels. This kit contains no actual virus or biohazardous material.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2006, 2007, 2010, 2011 EDVOTEK, Inc., all rights reserved EVT 2011_06_07AM
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BEFORE YOU START THE EXPERIMENT:
1. Read all instructions before starting the experiment.
2. If you will be conducting PCR using a thermal cycler without a heated lid, also read the Appendix entitled “Preparation and Handling PCR Samples with Wax”.
If you will be using three waterbaths to conduct PCR, read the two ap-pendices entitled “Polymerase Chain Reaction Using Three Waterbaths” and “Handling Samples with Wax Overlays”.
3. Writeahypothesisthatreflectstheexperimentandpredictexperimen-tal outcomes.
EXPERIMENT OBJECTIVE:
The objective of this experiment is to afford students the opportunity to understand the process of Real Time PCR and to learn the basic principles of DNA amplification by the Polymerase Chain Reaction.
BRIEF DESCRIPTION OF THE EXPERIMENT:
In this experiment, students will determine the amount of DNA in simulated patient samples, to determine levels (“titers”) of a viral infection. Here, small amounts of the PCR reaction will be removed after 0, 10, 20, 30, & 40 PCR cycles. Those reaction portions will be “spotted” on a Spot Plate and compared to standards containing known amounts of DNA. Rather than SYBR Green, DNA will be stained with ethidium bromide, a dye commonly used for detecting DNA in agarose gels. This kit contains no actual virus or biohazardous material.
Experiment Overview and General Instructions
9Real Time PCR
370EDVO-Kit #
TheBiotechnologyEducationCompany®•1-800-EDVOTEK•www.edvotek.com
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2006, 2007, 2010, 2011 EDVOTEK, Inc., all rights reserved EVT 2011_06_07AM
The Exp
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LABORATORY SAFETY
1. Gloves and goggles should be worn rou-tinely as good laboratory practice.
2. Exercise extreme caution when working with equipment that is used in conjunc-tion with the heating and/or melting of reagents.
3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.
4. Exercise caution when using any electrical equipment in the laboratory.
• Althoughelectricalcurrentfromthepowersourceisautomaticallydisrupted when the cover is removed from the apparatus, first turn off the power, then unplug the power source before disconnecting the leads and removing the cover.
• Turnoffpowerandunplugtheequipmentwhennotinuse.
5. EDVOTEK injection-molded electrophoresis units do not have glued junc-tions that can develop potential leaks. However, in the unlikely event that a leak develops in any electrophoresis apparatus you are using, IMMEDIATELY SHUT OFF POWER. Do not use the appara-tus.
6. Always wash hands thoroughly with soap and water after handling reagents or bio-logical materials in the laboratory.
Experiment Overview and General Instructions
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2006, 2007, 2010, 2011 EDVOTEK, Inc., all rights reserved EVT 2011_06_07AM
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Experimental Procedures
Module I: Preparation of the DNA Standard with Ethidium Bromide
Each student group should obtain 6 microcentrifuge tubes, each containing 6 µl of each of the DNA standard dilutions.
• ToeachtubecontainingtheDNAstandarddilution,add6µlofdiluteEthidiumBromide.Mix well and keep on ice.
Module II: Amplification of Template DNA
Each student group should obtain the following items from the instructor:
• OnetubecontainingaPCRreactionpellet™(componentA) • Onetubecontaining12µlofPrimermixture(componentB) • Onetubecontaining12µlofTemplateDNA(componentD) • Onetubecontaining25µlUltraPurewater(componentE)
1. Label five 0.5 ml tubes: “0” (Control), “10”, “20”, “30” and “40” along with your initials or group designation. These tubes will be used to collect PCR product at different time points.
2. Transfer the PCR reaction pellet™ to the appropriate sized tube (e.g. 0.5 ml or 0.2 ml) for your thermal cycler.
3. Label the tube containing the PCR reaction pellet™ with your initials or group designa-tion.
4. Tap the reaction tube to assure the reaction pellet is at the bottom of the tube.
5. Add the following to the pellet: Primer Mixture 10 µl Template DNA 10 µl Water 7 µl
6. Gently mix the PCR reaction tube and quickly spin it in a microcentrifuge to collect all the sample at the bottom of the tube. Make sure the PCR reaction pellet™ is completely dis-solved.
7. Remove 5 µl of reaction mix to a microcentrifuge tube labeled “0”. Keep the tube on ice.
8. Place PCR reaction tube in thermal cycler. Set PCR program as follows: 45 cycles of: 94°C x 45 sec 45°C x 45 sec 72°C x 45 sec Final Extension: 72°C x 5 min. Hold 4°C if necessary
11Real Time PCR
370EDVO-Kit #
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Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2006, 2007, 2010, 2011 EDVOTEK, Inc., all rights reserved EVT 2011_06_07AM
The Exp
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1.5 ng
6 ng
12.5 ng
25 ng
60 ng
600 ng
Figure A - Spot Standards Diagram
9. Sequentially remove 5 µl of PCR product and place in a labeled microcentri-fuge tube according to the following schedule.
Approximate Stop After Cycle Elapsed Time Add to Tube labeled 10 25 minutes “10” 20 50 minutes “20” 30 75 minutes “30” 40 100 minutes “40”
10. Keep samples on ice until all time point samples have been collected.
11. After the final sample has been collected, add 5 µl of Dilute Ethidium Bro-mide to each of the tubes containing the PCR product collected at different time points. Vortex to mix well.
12. Brieflycentrifugetocollectallsamplesatthebottomofthetubes.
13. In all of the following steps, use gloves to handle the Spot Plate.
14. Place the pre-cut Spot Plate on a paper towel.
15. Mark your initials or group designation on the very end of the Spot Plate. Using a micropipette, apply 50-60 µl of DNA Binding Solution to the top side of the plate. Use a paper towel or lint-free wipe to rub the solution over the surface of the entire plate.
16. Allow the Spot Plate to dry for about 2-3 minutes at room temperature.
17. Place the Spot Plate on the surface of a short /mid range UV transillumina-tor. (DO NOT turn the Transilluminator on now.)
18. Transfer 10 µl of each DNA standard dilution + Ethidium Bromide (from Module I) to the LEFT side of Spot Plate (see Figure A: Spot Plate standard diagram)
19. Transfer 10 µl of each PCR product + Ethidium Bromide (from Module II) to the RIGHT side of Spot Plate (see Figure A: Spot Plate standard diagram).
20. Switch ON UV Transilluminator and view results. Photograph and/or record observations.
Note: When transferring samples onto the Spot Plate (Step 18), try not to introduce air bubbles to the spots as it will interfere with the results.
Experimental Procedures
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2006, 2007, 2010, 2011 EDVOTEK, Inc., all rights reserved EVT 2011_06_07AM
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Answer the following study questions in your laboratory notebook or on a separate worksheet.
1. What is PCR? What are some of its applications?
2. Why is it often desirable to quantitate the amount of DNA in a particu-lar sample?
3. What is reverse transcription? What can it be used for?
4. What is the name of the dye used in quantitative PCR? How does that dye allow one to determine the amount of starting DNA in a sample?
Study Questions
Real Time PCR
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Mon - Fri 9 am - 6 pm ET
(1-800-338-6835)
EDVO-TECH SERVICE
1-800-EDVOTEK
Mon - Fri9:00 am to 6:00 pm ET
FAX: (301) 340-0582web: www.edvotek.com
email: info@edvotek.com
Please have the following information ready:
• Experiment number and title• Kit lot number on box or tube• Literature version number (in lower right corner)• Approximate purchase date
Technical ServiceDepartment
Instructor’s Guide
Class size, length of laboratory sessions, and availability of equipment are factors which must be considered in the planning and the implementation of this experiment with your students. These guidelines can be adapted to fit your specific set of circumstances. If you do not find the answers to your questions in this section, a variety of resources are continuously being added to the EDVOTEK web site. In addition, Technical Service is available from 9:00 am to 6:00 pm, Eastern time zone. Call for help from our knowledge-able technical staff at 1-800-EDVOTEK (1-800-338-6835).
NATIONAL CONTENT AND SKILL STANDARDS
By performing this experiment, students will learn to extract chromosomal DNA, load samples and run agarose gel electrophoresis. Analysis of the ex-periments will provide students the means to transform an abstract concept into a concrete explanation. Please visit our website for specific content and skill standards for various experiments.
EDUCATIONAL RESOURCES
Electrophoresis Hints, Help and Frequently Asked Questions
EDVOTEK Electrophoresis Experiments are easy to perform and are designed for maximum success in the classroom setting. However, even the most
experienced students and teachers occasionally encounter experimental problems or difficulties. The EDVOTEK web site provides several sug-gestions and reminders for conducting electro-phoresis, as well as answers to frequently asked electrophoresis questions.
Laboratory Extensions and Supplemental Activities
Laboratory extensions are easy to perform using EDVOTEK experiment kits. For laboratory exten-sion suggestions, please check the EDVOTEK website, which is updated on a continuous basis with educational activities and resources.
Visit our web site for information about
EDVOTEK's complete line of experiments for
biotechnology and biology education.
Online Ordering now available
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2006, 2007, 2010, 2011 EDVOTEK, Inc., all rights reserved EVT 2011_06_07AM
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PreLab Preparation
A. PREPARATION OF SERIAL DILUTIONS OF DNA STANDARD
1. Label six 0.5 ml microcentrifuge tubes 1 through 6 as shown below (tube 1 is the DNA standard i.e. component C)
2. Add the following volume of TE (component F) to tubes 2 through 6 as shown below.
180 ul in tube 2 120 ul in tube 3 187.5 ul in tube 4 100 ul in tube 5 152 ul in tube 6
3. Serially dilute the DNA standard by transferring the indicated volume from tube 1 through 6 as shown in figure below. Pipet the solution up and down or vortex to mix the solution well between each dilution.
4. You will now have the following concentration of DNA standard in each tube.
Tube 1: DNA standard [1µg/µl] = 1000 [ng/µl] (component C) Tube 2: [0.1 µg/µl] = [100 ng/µl] Tube 3: [0.04 µg/µl] = [40 ng/µl] Tube 4: [2.50 ng/µl] Tube 5: [1.25 ng/µl] Tube 6: [0.30 ng/µl]
5. From the dilutions prepared above, dispense 6 µl into properly labeled 0.5 ml microcentrifuge tubes. Each student group will receive 6 micro-centrifuge tubes, each containing 6 µl of each of the DNA standard dilu-tions.
This experiment isdesigned for 10 student groups.
20 µl 80 µl 12.5 µl 100 µl
180 µl TEDNA standard[1000 ng/µl)
120 µl TE 187.5 µl TE 100 µl TE
Dilution 1 Dilution 2 Dilution 3 Dilution 4
1 2 3 4 5
Dilution 5
48 µl
152 µl TE
6
Dilution 6
15Real Time PCR
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Instru
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Experiment Results and Analysis
1.5 ng
6 ng
12.5 ng
25 ng
60 ng
600 ng
Increasing cycle number should result in increasingEthidiumBromidefluorescence,asshown in the hypothetical outcome shown below.Itislikelythatat~30cycles,thefluo-rescence will be saturated, indicating that no more PCR product can be formed.
B. PREPARATION OF OTHER EXPERIMENTAL COMPONENTS
1. Label 10 microcentrifuge tubes “dilute EtBr”. Wear proper safety gear and dispense 100 µl of dilute Ethidium Bromide per tube. Distribute on tube per group.
2. Distribute one tube containing a PCR reaction pellet™ (component A) per group.
3. Label 10 microcentrifuge tubes “Primer”. Dispense 12 µl of Primer mix-ture (component B) per tube. Distribute one tube per group.
4. Label 10 microcentrifuge tubes “Template DNA”. Dispense 12 µl of Tem-plate DNA (component D) per tube. Distribute one tube per group.
5. Label 10 microcentrifuge tubes “Water”. Dispense 25 µl of UltraPure water (component E) per tube. Distribute one tube per group.
6. Distribute one spot plate for spotting DNA samples per group.
7. The DNA binding solution can be shared by all groups.
PreLab Preparation
Please refer to the kit insert for the Answers to
Study Questions
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Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2006, 2007, 2010, 2011 EDVOTEK, Inc., all rights reserved EVT 2011_06_07AM
Ap
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centrifuge tube to ensure direct contact with the reagents used in subse-quent steps.
4. Cheek Cells: A white pellet must be visible after centrifuging the cell sus-pension obtained from cheek cell swabbing. If necessary, repeat the centrif-ugation to obtain a visible pellet. After removal of the supernatant, suspend the pellet in the chelating agent by repeated vortexing and pipetting up and down.
5. Chelating Agent: Chelating agent removes Mg (required by DNA-degrading nucleases and DNA polymerases). The small beads must be suspended in the buffer prior to delivery to the cells (i.e., mix the chelating agent just before you transfer it to the tube containing the cells.
Appendix: PCR Experimental Success Guidelines
EDVOTEK experiments which involve the extraction and amplification of DNA for fingerprinting are extremely relevant, exciting and stimulating classroom laboratory activities. These experiments have been performed successfully in many classrooms across the country, but do require careful execu-tion because of the small volumes used. The following guidelines offer some important suggestions, reminders and hints for maximizing success.
DNA Extraction and Sample Preparation
CELL PREPARATION:
1. Sufficient Cells: It is critical that there are sufficient cells to obtain enough DNA that will yield posi-tive DNA fingerprinting results. Cell sources include human, plant, drosophila and bacterial cells. Without enough cells, there will not be enough DNA template for the PCR reaction.
2. Human (Self) DNA Fingerprinting: Cells obtained from human sources, such as cheek cells, need to be harvested cautiously. Aerosol can result and cross-contamination among students can be a health hazard. Hair follicles do not pose the same problem and yield sufficient DNA required for the PCR reaction.
3. Hair Cells: At least four (4) hair follicles are needed. The preferred source is hair from eyebrows. Use only hairs containing a sheath, a barrel-shaped structure (often white in color) encircling the shaft near the base of the hair (see figure at left). Centrifuge the hair follicles to the bottom of the micro-
6. Boiling: The boiling step for 10 minutes is required to obtain cell lysis. Boiling will not degrade the DNA and nucleases will NOT degrade DNA in the absence of Mg.
7. Centrifugation: Centrifuge the cell suspension carefully after cooling. If the pellet loosens, repeat this step. The supernatant should be clear, not cloudy, and the pellet should be solid at the bottom of the tube. Repeat centrifugation for a longer period of time, if necessary.
8. DNA Transfer: Transfer the DNA to a new microcentrifuge tube very carefully. It is the step prior to the PCR reaction. If any chelating agent beads (as few as one or two) are transferred, they can easily trap the Mg required by the Taq DNA polymerase as a cofactor for catalysis. As an additional precau-tion, centrifuge the supernatant a second time.
Remember: Any carry-over of chelating agent to the PCR reaction will not yield results.
Shaft
Sheath
Root
HUMANHAIR
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THE PCR REACTION
9. Add Primers and DNA to the PCR Reaction Bead: Add the primer mixture (forward and reverse prim-ers) and the cell DNA (supernatant) as specified in the experimental procedures to the microcentri-fuge tube containing the PCR reaction bead. Make sure that the bead (which contains the Taq DNA polymerase, the 4XdTPs, Mg and the PCR reaction buffer) is completely dissolved. Do a quick spin in a microcentrifuge to bring the entire sample to the bottom of the tube. Prepare the control reaction similarly.
10. The Thermal cycler: The thermal cycler must be programmed for the correct cycle sequence. It is criti-cal that the temperatures and the time for each of the cycles are accurate.
11. Oil or Wax: For certain thermal cyclers which do not have a top heating plate, it is necessary to over-lay the reaction in the microcentrifuge tubes with oil or wax to prevent evaporation.
12. Manual Water Bath PCR: Three water baths can be used as an alternative to using a thermal cycler for PCR. Samples require oil or wax layers. This method requires extra care and patience and results are more variable than when using a thermal cycler.
GEL PREPARATION AND STAINING
13. Concentrated agarose: Gels of higher concentration (> 0.8%) require special attention when dissolv-ing or re-melting. Make sure that the solution is completely clear of “clumps” or glassy granules. Distorted electrophoresis DNA band patterns will result if the gel is not properly prepared.
14. Electrophoretic separation: The tracking dye should travel at least 6 cm from the wells for adequate separation before staining.
15. Staining: Staining of higher concentration gels (> 0.8%) require additional care to obtain clear, visible results.
• Afterstaining(15to30min.)withInstaStain®EthidiumBromideorliquidethidiumbromide,examine the results using a UV (300nm) transilluminator. Repeat the staining as required.
• GelsstainedwithInstaStain®Blueorotherliquidbluestainmayfadewithtime.Re-stainthegelto visualize the DNA bands.
16. DNA 200 bp markers: After staining the agarose gel, the DNA 200 bp markers should be visible after staining. If there are visible bands in the markers and control lanes, but bands in the sample lanes are faint or absent, it is possible that the DNA was not successfully extracted from the cells. If markers, control and DNA bands are all faint or absent, problems could potentially be due to improper prepara-tion of the gel, absence of buffer in the gel, improper gel staining or a dysfunctional electrophoresis unit or power source.
Appendix: PCR Experimental Success Guidelines
19Real Time PCR
370EDVO-Kit #
TheBiotechnologyEducationCompany®•1-800-EDVOTEK•www.edvotek.com
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2006, 2007, 2010, 2011 EDVOTEK, Inc., all rights reserved EVT 2011_06_07AM
Ap
pen
dix
Appendix: Polymerase Chain Reaction Using Three Waterbaths
Superior PCR results are obtained using an automated thermal cycler. However, if you do not have a ther-mal cycler, this experiment can be adapted to use three waterbaths (Cat. # 544). Much more care needs to be taken when using the three-waterbath PCR method. The PCR incubation sample is small and can easily be evaporated. Results using three waterbaths are often variable.
PREPARATION OF THE PCR REACTION:
1. The PCR reaction sample should be prepared as specified in the experiment instructions. Each PCR reaction sample contains the following three critical components:
•PCRReactionpellet™ (Each pellet contains Taq DNA polymerase, four deoxytriphosphates, Mg+2 and buffer.) •Primermix •DNAforamplification
2. After adding the components of the PCR reaction sample, use clean forceps to transfer one wax bead to the PCR tube. At the start of the PCR reaction, the wax will melt and overlay the samples to pre-vent evaporation during heating.
POLYMERASE CHAIN REACTION CYCLING
3. The three-waterbath PCR method requires three separate waterbaths, each set at different tempera-tures. The PCR reaction sample is sequentially cycled between the three waterbaths for a specified period of time. The sequential placement of the reaction sample in the waterbaths maintained at three different temperatures constitutes one PCR cycle. A typical PCR cycle might be set up as fol-lows:
94°C for 45 seconds 45°C for 45 seconds 72°C for 45 seconds
It is imperative that the temperatures are accurately maintained throughout the experiment.
4. The PCR tube must be handled carefully when sequentially cycled between the three waterbaths. For each cycle:
• CarefullyplacethePCRtubeinawaterbathfloat.Makesurethatthesamplevolumeisatthebottomofthetubeandremainsundisturbed.Ifatubefallsonthelabbenchorfloor,pulsespinthe tube in a balanced microcentrifuge, or shake the tube to get all of the sample to the bottom of the tube.
• Useforcepstocarefullylowerthewaterbathfloat(withtubes)sequentiallyintothewaterbaths.
5. Process the PCR reaction sample for the total number of cycles specified in the experiment instruc-tions. On the final cycle the 72°C incubation can be extended to 5 minutes.
6. After all the cycles are completed, the PCR sample is prepared for electrophoresis.
Please refer to the Appendix entitled "PCR Samples with Wax Overlays" for sample handling and preparation tips.
Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 2006, 2007, 2010, 2011 EDVOTEK, Inc., all rights reserved EVT 2011_06_07AM
20
370Real Time PCR
TheBiotechnologyEducationCompany®•1-800-EDVOTEK•www.edvotek.com
EDVO-Kit #A
pp
end
ix
Appendix: Preparation and Handling of PCR Samples With Wax
For Thermal Cyclers without Heated Lids, or PCR Using Three Waterbaths
Automated thermal cyclers with heated lids are designed to surround the entire sample tube at the appro-priate temperature during PCR cycles. Heating the top of the tubes during these cycles prevents the very small sample volumes from evaporating. For thermal cyclers without heated lids, or when conducting PCR by the three-waterbath method, it is necessary to add a wax bead or pellet to the reaction sample. During the PCR process, the wax will melt and overlay the samples to prevent evaporation during heating.
PREPARING THE PCR REACTION:
1. The PCR reaction sample should be prepared as specified in the experiment instructions. Each PCR reaction sample contains the following three critical com-ponents:
•PCRReactionpellet™ (Each pellet contains Taq DNA polymerase,
four deoxytriphosphates, Mg+2 and buffer.)
• Primermix
• DNAforamplification
2. After adding the components of the PCR reaction sample, use clean forceps to transfer one wax bead to the PCR tube.
3. Process the PCR reaction sample for the total number of cycles specified in the experiment instructions.
PREPARING THE PCR REACTION FOR ELECTROPHORESIS:
4. After the cycles are completed, transfer the PCR tube to a rack and prepare the PCR sample for electrophoresis.
• PlacethePCRtubeina94°Cwaterbathlong enough to melt the wax overlay. Use a clean pipet to remove most of the melted wax overlay.
• Allowathinlayerofthewaxtosolidify.
• Useacleanpipettiptogentlypokeaholethrough the solidified wax. Remove the tip.
• Useanothercleanpipettiptoenterthehole to remove the volume of mixture specified in the experiment instructions. Transfer this volume to a clean tube.
• Addotherreagentsaccordingtoexperi-ment instructions, if applicable,.
• Add5µlof10xGelLoadingsolutiontothe sample and store on ice.
5. Proceed to delivery of the sample onto an agarose gel for electrophoresis as specified in the experiment instructions.
Mat
eria
l Saf
ety
Dat
a Sh
eet
May
be
use
d t
o c
om
ply
wit
h O
SHA
's H
azar
d C
om
mu
nic
atio
nSt
and
ard
. 29
CFR
191
0.12
00 S
tan
dar
d m
ust
be
con
sult
ed f
or
spec
ific
req
uir
emen
ts.
IDEN
TITY
(A
s U
sed
on
Lab
el a
nd
Lis
t)N
ote
: B
lan
k sp
aces
are
no
t p
erm
itte
d.
If a
ny
item
is n
ot
app
licab
le, o
r n
o in
form
atio
n is
ava
ilab
le, t
he
spac
e m
ust
b
e m
arke
d t
o in
dic
ate
that
.
Sect
ion
IM
anu
fact
ure
r's
Nam
e
Sect
ion
II -
Haz
ard
ou
s In
gre
die
nts
/Id
enti
fy In
form
atio
n
Emer
gen
cy T
elep
ho
ne
Nu
mb
er
Tele
ph
on
e N
um
ber
fo
r in
form
atio
n
Dat
e Pr
epar
ed
Sig
nat
ure
of
Prep
arer
(o
pti
on
al)
Ad
dre
ss (
Nu
mb
er, S
tree
t, C
ity,
Sta
te,
Zip
Co
de)
EDV
OTE
K, I
nc.
1467
6 R
oth
geb
Dri
veR
ock
ville
, MD
208
50
Haz
ard
ou
s C
om
po
nen
ts [
Spec
ific
C
hem
ical
Iden
tity
; C
om
mo
n N
ame(
s)]
O
SHA
PEL
AC
GIH
TLV
Oth
er L
imit
s R
eco
mm
end
ed%
(O
pti
on
al)
(301
) 25
1-59
90
(301
) 25
1-59
90
Bo
ilin
g P
oin
t
Sect
ion
III -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
s
Un
usu
al F
ire
and
Exp
losi
on
Haz
ard
s
Spec
ial F
ire
Fig
hti
ng
Pro
ced
ure
s
Vap
or
Pres
sure
(m
m H
g.)
Vap
or
Den
sity
(A
IR =
1)
Solu
bili
ty in
Wat
er
Ap
pea
ran
ce a
nd
Od
or
Sect
ion
IV -
Ph
ysic
al/C
hem
ical
Ch
arac
teri
stic
sFl
ash
Po
int
(Met
ho
d U
sed
)
Exti
ng
uis
hin
g M
edia
Flam
mab
le L
imit
sU
ELLE
L
Mel
tin
g P
oin
t
Evap
ora
tio
n R
ate
(Bu
tyl A
ceta
te =
1)
Spec
ific
Gra
vity
(H
0 =
1)
2
Ag
aro
se
06-0
6-11
This
pro
du
ct c
on
tain
s n
o h
azar
do
us
mat
eria
ls a
s d
efin
ed b
y th
e O
SHA
Haz
ard
Co
mm
un
icat
ion
Stan
dar
d.
CA
S #9
012-
36-6
For
1% s
olu
tio
n 1
94 F
N
o d
ata
N
o d
ata
No
dat
a
No
dat
a
No
dat
a
Inso
lub
le -
co
ld
W
hit
e p
ow
der
, no
od
or
N.D
. = N
o d
ata
No
dat
a
N
.D.
N.D
.
Wat
er s
pra
y, d
ry c
hem
ical
, car
bo
n d
ioxi
de,
hal
on
or
stan
dar
d f
oam
Poss
ible
fir
e h
azar
d w
hen
exp
ose
d t
o h
eat
or
flam
e
No
ne
ED
VO
TE
K®
Stab
ility
Sect
ion
V -
Rea
ctiv
ity
Dat
aU
nst
able
Sect
ion
VI -
Hea
lth
Haz
ard
Dat
a
Inco
mp
atib
ility
Co
nd
itio
ns
to A
void
Ro
ute
(s)
of
Entr
y:In
hal
atio
n?
Ing
esti
on
?Sk
in?
Oth
er
Stab
le
Haz
ard
ou
s Po
lym
eriz
atio
nM
ay O
ccu
rC
on
dit
ion
s to
Avo
id
Will
No
t O
ccu
r
Hea
lth
Haz
ard
s (A
cute
an
d C
hro
nic
)
Car
cin
og
enic
ity:
NTP
?O
SHA
Reg
ula
tio
n?
IAR
C M
on
og
rap
hs?
Sig
ns
and
Sym
pto
ms
of
Exp
osu
re
Med
ical
Co
nd
itio
ns
Gen
eral
ly A
gg
rava
ted
by
Exp
osu
re
Emer
gen
cy F
irst
Aid
Pro
ced
ure
s
Sect
ion
VII
- Pr
ecau
tio
ns
for
Safe
Han
dlin
g a
nd
Use
Step
s to
be
Take
n in
cas
e M
ater
ial i
s R
elea
sed
fo
r Sp
illed
Was
te D
isp
osa
l Met
ho
d
Prec
auti
on
s to
be
Take
n in
Han
dlin
g a
nd
Sto
rin
g
Oth
er P
reca
uti
on
s
Sect
ion
VIII
- C
on
tro
l Mea
sure
s
Ven
tila
tio
nLo
cal E
xhau
stSp
ecia
l
Mec
han
ical
G
en. d
iluti
on
ven
tila
tio
n
Res
pir
ato
ry P
rote
ctio
n (
Spec
ify
Typ
e)
Pro
tect
ive
Glo
ves
Oth
er P
rote
ctiv
e C
loth
ing
or
Equ
ipm
ent
Wo
rk/H
ygie
nic
Pra
ctic
es
Eye
Pro
tect
ion
Haz
ard
ou
s D
eco
mp
osi
tio
n o
r B
ypro
du
cts
Yes
Sp
lash
pro
of
go
gg
les
Imp
ervi
ou
s cl
oth
ing
to
pre
ven
t sk
in c
on
tact
No
neX
N
on
e
No
dat
a av
aila
ble
X
No
ne
Yes
Y
es
Yes
Inh
alat
ion
: N
o d
ata
avai
lab
le
In
ges
tio
n:
Larg
e am
ou
nts
may
cau
se d
iarr
hea
No
dat
a av
aila
ble
No
dat
a av
aila
ble
Trea
t sy
mp
tom
atic
ally
an
d s
up
po
rtiv
ely
Swee
p u
p a
nd
pla
ce in
su
itab
le c
on
tain
er f
or
dis
po
sal
No
rmal
so
lid w
aste
dis
po
sal
No
ne
No
ne
Ch
emic
al c
artr
idg
e re
spir
ato
r w
ith
fu
ll fa
cep
iece
.
Mat
eria
l Saf
ety
Dat
a Sh
eet
May
be
used
to c
ompl
y w
ith O
SHA'
s H
azar
d C
omm
unic
atio
nSt
anda
rd.
29 C
FR 1
910.
1200
Sta
ndar
d m
ust b
e co
nsul
ted
for
spec
ific
requ
irem
ents
.
IDEN
TITY
(As
Use
d on
Lab
el a
nd L
ist)
Not
e: B
lank
spa
ces
are
not p
erm
itted
. If
any
item
is n
ot
appl
icab
le, o
r no
info
rmat
ion
is a
vaila
ble,
the
spac
e m
ust
be m
arke
d to
indi
cate
that
.
Sect
ion
IM
anuf
actu
rer's
Nam
e
Sect
ion
II - H
azar
dous
Ingr
edie
nts/
Iden
tify
Info
rmat
ion
Emer
genc
y Te
leph
one
Num
ber
Tele
phon
e N
umbe
r for
info
rmat
ion
Dat
e Pr
epar
ed
Sign
atur
e of
Pre
pare
r (op
tiona
l)
Addr
ess
(Num
ber,
Stre
et, C
ity, S
tate
, Zi
p C
ode)
EDVO
TEK
, Inc
.
1467
6 R
othg
eb D
rive
Roc
kvill
e, M
D 2
0850
Haz
ardo
us C
ompo
nent
s [S
peci
fic
Che
mic
al Id
entit
y; C
omm
on N
ame(
s)]
O
SHA
PEL
ACG
IH T
LVO
ther
Lim
its
Rec
omm
ende
d%
(Opt
iona
l)
(301
) 251
-599
0
(301
) 251
-599
0
Boilin
g Po
int
Sect
ion
III -
Phys
ical
/Che
mic
al C
hara
cter
istic
s
Unu
sual
Fire
and
Exp
losi
on H
azar
ds
Spec
ial F
ire F
ight
ing
Proc
edur
es
Vapo
r Pre
ssur
e (m
m H
g.)
Vapo
r Den
sity
(AIR
= 1
)
Solu
bilit
y in
Wat
er
Appe
aran
ce a
nd O
dor
Sect
ion
IV -
Phys
ical
/Che
mic
al C
hara
cter
istic
sFl
ash
Poin
t (M
etho
d U
sed)
Extin
guis
hing
Med
ia
Flam
mab
le L
imits
UEL
LEL
Mel
ting
Poin
t
Evap
orat
ion
Rat
e(B
utyl
Ace
tate
= 1
)
Spec
ific
Gra
vity
(H 0
= 1
) 2
Ethi
dium
Bro
mid
e
Ethi
dium
Bro
mid
e
Dat
a no
t ava
ilabl
e
No
data
No
data
No
data
No
data
No
data
No
data
Solu
ble
liqui
d, n
o od
or
No
data
N.D
. = N
o da
ta
N.D
.
N.D
.
Wat
er sp
ray,
car
bon
diox
ide,
dry
che
mic
al p
owde
r or a
ppro
pria
te fo
am
Wea
r sel
f-co
ntai
ned
brea
thin
g ap
para
tus a
nd p
rote
ctiv
e cl
othi
ng to
prev
ent c
onta
ct w
ith sk
in a
nd e
yes.
Emits
toxi
c fu
mes
und
er fi
re c
ondi
tions
CA
S# 1
239-
45-8
Stab
ility
Sect
ion
V - R
eact
ivity
Dat
aU
nsta
ble
Sect
ion
VI -
Hea
lth H
azar
d D
ata
Inco
mpa
tibilit
y
Con
ditio
ns to
Avo
id
Rou
te(s
) of E
ntry
:In
hala
tion?
Inge
stio
n?Sk
in?
Oth
er
Stab
le
Haz
ardo
us
Poly
mer
izat
ion
May
Occ
urC
ondi
tions
to A
void
Will
Not
Occ
ur
Hea
lth H
azar
ds (A
cute
and
Chr
onic
)
Car
cino
geni
city
:N
TP?
OSH
A R
egul
atio
n?IA
RC
Mon
ogra
phs?
Sign
s an
d Sy
mpt
oms
of E
xpos
ure
Med
ical
Con
ditio
ns G
ener
ally
Agg
rava
ted
by E
xpos
ure
Emer
genc
y Fi
rst A
id P
roce
dure
s
Sect
ion
VII -
Pre
caut
ions
for S
afe
Han
dlin
g an
d U
seSt
eps
to b
e Ta
ken
in c
ase
Mat
eria
l is
Rel
ease
d fo
r Spi
lled
Was
te D
ispo
sal M
etho
d
Prec
autio
ns to
be
Take
n in
Han
dlin
g an
d St
orin
g
Oth
er P
reca
utio
ns
Sect
ion
VIII
- Con
trol
Mea
sure
s
Vent
ilatio
nLo
cal E
xhau
stSp
ecia
l
Mec
hani
cal (
Gen
eral
)
Res
pira
tory
Pro
tect
ion
(Spe
cify
Typ
e)
Prot
ectiv
e G
love
s
Oth
er P
rote
ctiv
e C
loth
ing
or E
quip
men
t
Wor
k/H
ygie
nic
Prac
tices
Eye
Prot
ectio
n
Haz
ardo
us D
ecom
posi
tion
or B
ypro
duct
s
X
N
one
Stro
ng o
xidi
zing
age
nts
Car
bon
mon
oxid
e, C
arbo
n di
oxid
e, n
itrog
en o
xide
s, h
ydro
gen
brom
ide
gas
X
N
one
Yes
Yes
Ye
s
No
data
ava
ilabl
e
with
eye
s & sk
in, r
inse
imm
edia
tely
with
cop
ious
am
ount
s of w
ater
for 1
5 m
inut
es.
If in
hale
d, re
mov
e to
fr
esh
air.
If sw
allo
wed
, was
h ou
t mou
th w
ith w
ater
& se
ek m
edic
al a
dvic
e. W
ash
cont
amin
ated
clo
thin
g.
No
data
In c
ase
of a
ccid
ent s
eek
med
ical
adv
ice
imm
edia
tely
. In
cas
e of
con
tact
Wea
r res
pira
tor,
chem
safe
ty g
oggl
es, b
oots
and
hea
vy g
love
s. M
op u
p, p
lace
in b
ad a
nd h
old
for w
aste
disp
osal
. Av
oid
inha
ling.
Ven
tilat
e ar
e an
d w
ash
spill
site
. M
ix m
ater
ial w
ith c
ombu
stib
le so
lven
t and
bur
n in
a c
hem
ical
inci
nera
tor
equi
pped
afte
rbur
ner a
nd sc
rubb
er
Use
in c
hem
ical
fum
e ho
od w
ith p
rope
r pro
tect
ive
lab
gear
.
Mut
agen
Yes
Che
m. f
ume
hood
No
Non
e
chem
ical
resi
stan
t glo
ves
Che
m. s
afet
y go
ggle
s
Rub
ber b
oots
Use
in c
hem
ical
fum
e ho
od w
ith p
rope
r pro
tect
ive
lab
gear
.
Acut
e: M
ater
ial i
rrita
ting
to m
ucou
s m
embr
anes
, upp
er re
spira
tory
trac
t, ey
es, s
kin
Chr
onic
: M
ay c
ause
her
itabl
e ge
netic
dam
age.
NIO
SH/M
SHA
app
rove
d re
spira
tor
irrita
ting
to e
yes,
resp
irato
ry sy
stem
and
skin
06-0
6-11
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OTE
K®
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