Real Time PCR - EDVOTEK · as real time PCR and uses an instrument that couples PCR with...

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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

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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.

http://www.edvotek.com

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EDVOTEK - The Biotechnology Education Company® 1-800-EDVOTEK • www.edvotek.com

FAX: (301) 340-0582 • email: [email protected]

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


mailto:[email protected]

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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TheBiotechnologyEducationCompany®•1-800-EDVOTEK•www.edvotek.com

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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.


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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.


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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.



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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


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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



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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


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1.5 ng

6 ng

12.5 ng

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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



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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


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EDVOTEK - The Biotechnology Education Company® 1-800-EDVOTEK • www.edvotek.com

FAX: (301) 340-0582 • email: [email protected]

370EDVO-Kit #

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: [email protected]

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






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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


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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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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.

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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


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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.



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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.


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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

EDV

OTE

K®

Real Time PCR - EDVOTEK · as real time PCR and uses an instrument that couples PCR with...

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Transcript of Real Time PCR - EDVOTEK · as real time PCR and uses an instrument that couples PCR with...