1.
One-step RT-PCR
Kit for Virus
Detection
For fast and sensitive virus one-step
RT-PCR
www.tiangen.com/en
SD121221
One-step RT-PCR Kit for Virus
Detection
Cat. no. SD102
Kit Contents
Contents
SD102
50 µLx50 rxn
Hotmaster Taq Polymerase (2.5 U/μl)
130 μl
Quant RTase (for one step)
30 μl
RNasin (40 U/μl)
30 μl
10x sd RT-PCR Buffer
300 μl
Super Pure dNTP
(10 mM each)
120 μl
RNase-Free ddH2O
2 × 1 ml
5x RT-PCR Enhancer
600 μl
Handbook
1
Storage
One-step RT-PCR Kit for Virus Detection should be stored at -20 °C.
Introduction
RT-PCR is the technology by which RNA template is reversely
transcribed to cDNA by the transcriptase and then target fragment
is obtained by amplification using PCR based on the cDNA template.
RT-PCR is applied in detection of gene expression level in cells,
tissues, virus and cloning the cDNA of the specific gene.
The One-step RT-PCR Kit for Virus Detection contains a specially
formulated enzyme blend for both reverse transcription and PCR.
Only one reaction mix needs to be set up: no additional reagents
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need to be added after the reaction starts, which avoids
contaminations and enhances sensitivity of detection.
Although all of the enzymes are present in the reaction mix, the use
of Hotmaster Taq polymerase ensures the temporal separation of
reverse transcription and PCR, allowing both processes to be
performed sequentially in a single tube. One-step RT-PCR Kit for
Virus Detection contains unique reaction buffers which ensure the
high enzymatic activity of the Quant RTase and Hotmaster Taq
polymerase.
Materials required but not supplied
1.
RNA templates
2.
gene-specific PCR primers
Important Notes
1.
RNA template could be total RNA or mRNA. SD101 TIANamp
RNA Kit for Virus Detection is recommended for extraction of
high-purity RNA.
2.
For some low abundance gene and complex gene, addition of
0.5 μM oligo (dT) to 50 μL system may lead to high
amplification efficiency.
3.
For high-GC and low abundance gene or complex gene,
adjusting the reverse transcription temperature may lead to
high amplification efficiency.
4.
RNase contaminations should be avoided in one-step RT-PCR.
Some measures can be taken as below:
1)
Wear a disposable gloves and respirator to avoid the RNase
contaminations from skin and saliva
2)
Operate the RNA related experiments in an RNase-Free
environment using RNase-Free
apparatus and consumable
items.
3)
Consumable items related with RT-PCR should be incubated in
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0.1% DEPC solution at 37°C for 12 hours and sterilized for 30
min before use.
5.
Quant RTase, RNasin, and Hotmaster Taq polymerase should
be centrifuged briefly before use. Pipet slowly and store at
-20°C immediately after use.
6.
Repeated freezing and thawing of dNTP should be avoided,
since this leads to losing effectiveness
7.
Only use gene-specific primers and use specific primers
according to specific requirements of experiments.
8.
Make sure the thermal cycler is preheated to 50°C before
placing samples in it.
9.
The primers quality affects RT-PCR result. GC abundance,
primer binding site and length of primers should be
considered. Professional software is recommended for primer
design.
Protocol
1.
Thoroughly thaw the template RNA, primer solutions, Super
pure dNTP Mixture, 10 × sdRT-PCR Buffer, RNase-Free ddH2O
and 5 × RT-PCR enhancer, centrifuge briefly and place them on
ice.
2.
Prepare a reaction solution according to the following table
Contents
Volume/Reaction
10 × sdRT-PCR Buffer
Super pure dNTP Mixture
(10 mM each)
5 µl
5× RT-PCR enhancer
10 µl
RNasin (40 U/µl)
0.5 µl
Hotmaster Taq polymerase
(2.5 U/µl)
2.5 µl
2 µl
One-step RT-PCR Kit for Virus Detection Handbook |
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Quant RTase (for one step)
0.5 µl
Forward Primer (10 µM)
Reverse Primer (10 µM)
3 µl
3 µl
Template RNA
10 ng-1 µg total RNA
RNase-Free ddH2O
Up to 50 µl
Notes: If setting up more than one RT-PCR reaction, mix all
components one time and divide into each tube.
3.
Start the RT-PCR program while PCR tubes are still on ice. Wait
until the thermal cycler has reached 50°C. Then place the PCR
tubes in the thermal cycler.
4.
Set up thermal cycler conditions according to the following
table.
Steps
Reaction
Time
Temperature
1
Reverse transcription
30 min
50°C
2
Initial denaturation
2 min
94°C
3
Denaturation
0.5-1 min
94°C
4
Annealing
0.5-1 min
50-60°C
5
Extension
0.5-2 min
65°C
6
7
5.
30-40 cycles from step 3 to step 5
Final extension
10 min
65°C
Analyze the PCR products using agarose gel electrophoresis.
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Ordering Information
RNA Isolation
Product
Size
Cat. no.
TIANamp Virus RNA Kit
50 preps
DP315-R
RNAprep Pure Kit (For Cell/Bacteria)
50 preps
DP430
RNAprep Pure Kit (For Tissue)
50 preps
DP431
RNAprep Pure Kit (For Plant)
50 preps
DP432
Product
Size
Cat. no.
DNA Marker I
50 lanes
MD101-01
200 lanes
MD101-02
50 lanes
MD103-01
200 lanes
MD103-02
50 lanes
MD108-01
200 lanes
MD108-02
DNA Marker
DNA Marker III
50 bp DNA Ladder
One-step RT-PCR Kit for Virus Detection Handbook |
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