Download HiPer® Real-Time PCR Teaching Kit

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 -20oC
Other kit contents can be stored at room temperature (15-25oC)
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 (λmax = 488 nm) and it emits green light (λmax = 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.
SYBR Green
Forward Primer
SYBR Green
Forward Primer
I
Excitation
II
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.
Product Code
No.
Materials Provided
Quantity
Storage
10 expts
1
MBT074*
Hi-SYBr Master Mix
0.240 ml
-20oC
2
TKC394*
Primer Mix
0.050 ml
-20oC
3
ML065
1 ml
-20oC
4
TKC395*
0.015 ml
-20oC
5
CG282
22 No
RT
Molecular Biology Grade Water for RT-PCR
Template DNA
Polypropylene Tubes, 0.2 ml (PCR Tubes)
* 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 -20oC. Other reagents can be stored at room temperature (15-25oC).
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
Add 10µl Hi-SYBr master mix in a PCR tube
and label it as “+ve”
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
make the final volume to 20 µl
Add 8 µl of Molecular Biology Grade to
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
Place the tubes in real-time PCR machine
and set the recommended PCR program
and set the recommended PCR program
Initial denaturation at 95oC for 10 minutes
Denaturation at 95oC for 45seconds
Annealing at 56oC for 30 seconds
Extension at 72oC for 30 seconds
Final Extension at 72oC for 10 minutes
6
27 cycles
Observation and Result:
Analyze the data from the amplification plot and observe the Ct values:
Sr.
No.
1
2
3
Sample
Negative control
1 µl of template DNA (amplicon
of E. coli O157:H7)
1 µl of template (amplicon of
E. coli O157:H7)
Ct
value
N/A
13.36
13.50
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.
1.
Problem
Solution
Cause
Degraded samples
No amplification
Error in protocol setup
2.
Variability
between
replicates
Error in reaction set-up
Air bubbles in reaction mix
Pipetting error
3.
Amplification
negative control
in
Reagents contaminated
Check the integrity of DNA using agarose gel
electrophoresis.
Verify that the correct reagent volumes, dilutions and
storage conditions have been used.
Prepare large volume master mix, vortex thoroughly
and aliquot into reaction tubes.
Briefly centrifuge reaction samples/plate prior to
running on a real-time PCR instrument.
Ct values of replicates can show increased variation
due to poor laboratory technique or imprecise
pipettes.
Repeat the analysis.
Technical Assistance:
At HiMedia we pride ourselves on the quality and availability of our technical support. For any kind of Technical
assistance mail at [email protected]
PIHTBM032 _O/0814
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HTBM032-00