HiPer Real-Time PCR Teaching Kit Product Code: HTBM032 Number of experiments that can be performed: 10 Duration of Experiment Protocol: 1.5 hours Storage Instructions: The kit is stable for 12 months from the date of receipt Store Hi-SYBr Master Mix, Primer Mix, Molecular Biology Grade Water and Template DNA at -20 o C Other kit contents can be stored at room temperature (15-25 o C) 1
Index Sr. No. Contents Page No. 1 Aim 3 2 Introduction 3 3 Principle 3 4 Kit Contents 5 5 Materials Required But Not Provided 5 6 Storage 5 7 Important Instructions 5 8 Procedure 6 9 Observation and Result 7 10 Interpretation 7 11 Troubleshooting Guide 7 2
Aim: To learn the process of Real-time PCR. Introduction: Real-time polymerase chain reaction is a molecular biology based laboratory technique for amplification and simultaneous quantification of a targeted DNA. This specialized reaction follows the principles of regular PCR in real time instead of detecting the end product at the end. For this reason it is called quantitative PCR which identifies the products using either a fluorescent dye or a probe. Principle: Polymerase Chain Reaction (PCR) is a very sensitive and specific method to amplify a precise fragment of DNA from a complex mixture of starting material called as template DNA. The three steps of a successful PCR reaction include Denaturation, Annealing and Extension which consists of a series of temperature changes that are repeated 25 40 times and performed in a specialized instrument called thermal cycler. The Real-time PCR or Quantitative PCR is a modified approach compared to standard PCR, where the amplified DNA is detected as the reaction progresses instead of detection at the end. Quantitative PCR is carried out in a thermal cycler with the capacity to illuminate each sample with a beam of light of a specified wavelength and detect the fluorescence emitted by the excited fluorophore. There are two methods for the detection of amplified products in Real-time PCR: 1. Using non-specific fluorescent dyes SYBR Green is a double-stranded (ds) DNA binding fluorescent dye which is excited using blue light (λ max = 488 nm) and it emits green light (λ max = 522 nm). It intercalates to ds DNA during PCR which results in fluorescence. Therefore, an increase in DNA product during PCR leads to an increase in fluorescence intensity that can be measured for each cycle and hence, DNA concentrations are quantified. However, SYBR Green binds to all dsdna PCR products which include nonspecific PCR products like primer-dimer. This can potentially interfere with, or prevent, accurate quantification of the intended target sequence. Forward Primer SYBR Green Forward Primer SYBR Green I II Excitation Polymerization DNA Polymerase Emission III IV Fig 1: The sequence of reactions during SYBR Green-based Real-Time PCR At the beginning of PCR, the reaction mixture contains denatured DNA, primers, and SYBR Green dye. The unbound dye molecules give a weak fluorescence that produces a minimal background signal which is subtracted during data analysis. During the elongation step, more and more dye molecules intercalate to the newly synthesized DNA. An increase in fluorescence signal is observed in real-time by a constant 3
monitoring of the reaction. When the DNA is denatured during the next heating cycle, the dye molecules are released and the fluorescence signal decreases. 2. Using sequence-specific Hydrolysis (TaqMan) DNA probes In this real-time PCR a sequencespecific oligonucleotide is labeled with a fluorescent reporter and allows detection when it hybridizes to its complementary sequence. Fluorescent reporter probes detect only the DNA containing the probe sequence; therefore, use of the reporter probe significantly increases specificity, and enables quantification even in the presence of non-specific DNA amplification. Fig 2: The sequence of reactions during Hydrolysis (TaqMan) based Real-Time PCR In this real-time PCR the fluorescent reporter is tagged at one end of the probe and a quencher of fluorescence at the opposite end. The fluorescence is prevented when the reporter comes in close proximity to the quencher. During the annealing and extension steps the probe hybridizes to the target, and the dsdna-specific 5' 3' exonuclease activity of Taq cleaves off the reporter. As a result, the reporter is separated from the quencher which generates fluorescence signal that is proportional to the amount of amplified product in the sample. The HiPer Real-time PCR Teaching Kit is designed for detection of specific sequence of a specific gene (450 bp) for E.coli O157 using SYBR Green based quantitative PCR technique. 4
Kit Contents: Table 1: Enlists the materials provided in this kit with their quantity and recommended storage. Sr. Quantity Product Code Materials Provided No. 10 expts Storage 1 MBT074* Hi-SYBr Master Mix 0.240 ml -20 o C 2 TKC394* Primer Mix 0.050 ml -20 o C 3 ML065 Molecular Biology Grade Water for RT-PCR 1 ml -20 o C 4 TKC395* Template DNA 0.015 ml -20 o C 5 CG282 Polypropylene Tubes, 0.2 ml (PCR Tubes) 22 No R T * Always give a quick spin before opening the vial as the liquid material may stick to the wall or to the cap of the vial Materials Required But Not Provided: Real-time Thermocycler, Vortex Mixer, Centrifuge, Micropipettes, Tips (preferably barrier), Crushed ice Storage: HiPer Real-time PCR Teaching Kit is stable for 12 months from the date of receipt without showing any reduction in performance. On receipt, store Hi-SYBr Master Mix, Primer Mix, Molecular Biology Grade Water and template DNA at -20 o C. Other reagents can be stored at room temperature (15-25 o C). Important Instructions: Before use all PCR components should be completely thawed on ice. Perform the amplification reactions in a clean area, preferably in a biosafety cabinet. Use of aerosol barrier pipette tips is recommended to reduce contamination risks from extraneous DNA templates. 5
Procedure: Flow Chart for setting up PCR Reaction Add 10µl Hi-SYBr master mix in a PCR tube and label it as +ve Add 10µl Hi-SYBr master mix in a PCR tube and label it as -ve Add 2 µl the Primer mix Add 2 µl the Primer mix Add 1µl of template DNA Add 7 µl of Molecular Biology Grade Water Add 8 µl of Molecular Biology Grade to make the final volume to 20 µl Water to make the final volume to 20 µl Centrifuge the tube briefly at 6000 rpm for about 10 seconds Centrifuge the tube briefly at 6000 rpm for about 10 seconds Place the tubes in real-time PCR machine and set the recommended PCR program Place the tubes in real-time PCR machine and set the recommended PCR program Initial denaturation at 95 o C for 10 minutes Denaturation at 95 o C for 45seconds Annealing at 56 o C for 30 seconds 27 cycles Extension at 72 o C for 30 seconds Final Extension at 72 o C for 10 minutes 6
Observation and Result: Analyze the data from the amplification plot and observe the Ct values: Sr. No. Sample C t value 1 Negative control N/A 2 1 µl of template DNA (amplicon 13.36 of E. coli O157:H7) 3 1 µl of template (amplicon of E. coli O157:H7) 13.50 Fig 3: Data representing real-time amplification of E. coli O157:H7 with C t values (provided in table) Interpretation: After performing the real-time PCR amplification of E. coli O157:H7 DNA one can analyze the data from the amplification plot and obtain the C t value. Troubleshooting Guide: Sr.No. Problem Solution Cause 1. No amplification Degraded samples Check the integrity of DNA using agarose gel electrophoresis. Error in protocol setup Verify that the correct reagent volumes, dilutions and storage conditions have been used. 2. Variability between Error in reaction set-up Prepare large volume master mix, vortex thoroughly and aliquot into reaction tubes. replicates Air bubbles in reaction mix Briefly centrifuge reaction samples/plate prior to running on a real-time PCR instrument. Pipetting error C t values of replicates can show increased variation due to poor laboratory technique or imprecise pipettes. 3. Amplification in Reagents contaminated Repeat the analysis. negative control Technical Assistance: At HiMedia we pride ourselves on the quality and availability of our technical support. For any kind of Technical assistance mail at mb@himedialabs.com PIHTBM032 _O/0814 HTBM032-00 7