1

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 manufacture

Store Hi-SYBr Master Mix, Primer Mix, Molecular Biology Grade Water

and Template DNA at -20oC

Other kit contents can be stored at room temperature (15-25oC)

HiPer® Real-Time PCR Teaching Kit

P r o d u c t I n f o r m a t i o n

The information contained herein is believed to be accurate and complete. However no warranty or guarantee whatsoever is madeor is to be implied with respect to such information or with respect to any product, method or apparatus referred to herein

Tel: 00-91-22-6147 1919Fax: 6147 1920, 2500 5764

Email : info@himedialabs.comWeb : www.himedialabs.com

A-516, Swastik Disha Business Park, Via Vadhani Indl. Est., LBS Marg, Mumbai - 400 086, India

23, Vadhani Industrial Estate,LBS Marg, Mumbai - 400 086, India. Tel. : (022) 4017 9797 / 2500 1607 Fax : (022) 2500 2286

Commercial Office Registered Office :

WHO GMP

CERTIFIED

15

Unz i p p i n g G en e s

2

Index

Sr. No. Contents Page No.

1 Aim 3

2 Introduction 3

3 Principle 3

4 Kit Contents 4

5 Materials Required But Not Provided 5

6 Storage 5

7 Important Instructions 5

8 Procedure 5

9 Observation and Result 6

10 Interpretation 6

11 Troubleshooting Guide 6

3

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 dyewhich is excited using blue light (λmax = 488 nm) and it emits green light (λmax = 522 nm). It intercalates tods DNA during PCR which results in fluorescence. Therefore, an increase in DNA product during PCR leadsto an increase in fluorescence intensity that can be measured for each cycle and hence, DNA concentrationsare quantified. However, SYBR Green binds to all dsDNA PCR products which include nonspecific PCRproducts like primer-dimer. This can potentially interfere with, or prevent, accurate quantification of theintended target sequence.

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 monitoring of the reaction. When the DNA is denatured during the next heating cycle, the dye molecules are released and the fluorescence signal decreases.

DNA Polymerase

Forward Primer SYBR Green

Forward Primer

SYBR Green

I II

III

Excitation Polymerization

Emission

IV

4

2. Using sequence-specific Hydrolysis (TaqMan) DNA probes – In this real-time PCR a sequence-specific oligonucleotide is labeled with a fluorescent reporter and allows detection when it hybridizes toits complementary sequence. Fluorescent reporter probes detect only the DNA containing the probesequence; therefore, use of the reporter probe significantly increases specificity, and enablesquantification 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.

Kit Contents:

Table 1: Enlists the materials provided in this kit with their quantity and recommended storage.

Sr.

No. Product Code Materials Provided

Quantity Storage

10 expts

1 MBT074* Hi-SYBr Master Mix 0.250 ml -20oC

2 TKC394* Primer Mix 0.050 ml -20oC

3 ML065 Molecular Biology Grade Water for PCR 1 ml -20oC

4 TKC395* Template DNA 0.015 ml -20oC

5 CG282 Polypropylene Tubes, 0.2 ml (PCR Tubes) 20 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 thevial

5

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.

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 manufacture without showing any reduction in performance. On receipt, store Hi-SYBr Master Mix, Primer Mix, Molecular Biology Grade Water and template DNA at -20oC. Other reagents can be stored at room temperature (15-25oC).

Important Instructions:

Procedure:

6

Observation and Result:

Analyze the data from the amplification plot and observe the Ct values:

Fig 3: Data representing real-time amplification of E. coli O157:H7 with Ct 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 Ct value.

Troubleshooting Guide:

Sr. No.

Sample Ct

value 1 Negative control N/A 2 1 μl of template DNA (amplicon

of E. coli O157:H7) 13.36

3 1 μl of template (amplicon of E. coli O157:H7)

13.50

Sr.No. Problem Cause

Solution

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 replicates

Error in reaction set-up Prepare large volume of master mix, vortex thoroughly and aliquot into reaction tubes.

Air bubbles in reaction mix Briefly centrifuge reaction samples/plate prior to running on a real-time PCR instrument.

Pipetting error Ct values of replicates can show increased variation due to poor laboratory technique or imprecise pipettes.

3. Amplification in negative control

Reagents contaminated Repeat the analysis.

7

PIHTBM032 _O/0419 HTBM032-01

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

Do not use if package is damaged

Storage temperature

15°C

25°C

HiMedia Laboratories Pvt. Limited, 23 Vadhani Industrial Estate, LBS Marg,Mumbai-86,MS,India