Ezmedtest Covid-19 One-Step qRT-PCR Kit

Ezmedtest Covid-19 One-Step qRT-PCR Kit

Cat. No. SM307-0100 – Size: 100 Reactions – Store at -20°C

Description

The Ezmedtest Covid-19 One-Step qRT-PCR Kit are compatible with TaqMan probes. The Kit provides a convenient, sensitive, and reproducible method to detect and quantify the RNA molecules by quantitative reverse transcription polymerase chain reaction (qRT -PCR). The components for cDNA synthesis, PCR amplification, and quantification are combined in a kit, using gene-specific primers, probes, and target RNAs from either total RNA or mRNA. The kit consists of three major components: RT/ HotStar Taq Mix, 2X Reaction Mix, and 50 mM Magnesium Sulfate (MgSO4). The RT/ HotStar Taq Mix contain a mixture of Reverse Transcriptase (RTase) and HotStar Taq DNA polymerase for optimal cDNA synthesis and PCR amplification.

The RTase is modified from the Moloney Murine Leukemia Virus (M-MLV) RTase, engineered to reduce RNase H activity and increase thermal stability. The HotStar Taq DNA polymerase is Taq DNA polymerase complexes with a proprietary antibody that blocks activity at ambient temperatures. Activity is restored after the enzyme activation step at 95°C, thereby providing an automatic “hot-start” for Taq DNA polymerase in PCR, increasing the sensitivity and specificity of PCR reaction. The 2X Reaction Mix consists of 6 mM MgSO4, deoxyribonucleotide triphosphates (dNTPs), and stabilizers provide a proprietary buffer system optimized for reverse transcription and PCR amplification.

One addition tube of 50 mM MgSO4 is included in the kit. For COVID-19 detection, the optimal concentration is 4 mM. An additional 0.5 µl for each reaction is recommended.

Features

  • Time efficiency – included most reagents for qRT-PCR reaction.

Applications

Detection of expressed genes. Examination of transcript variants.
Kit Contents
Contents SM307-0100
RT/ HotStar Taq Mix 50 μl
2X Reaction Mix 1.25 ml
50 mM MgSO4 200 μl

Quality Control

The quality of the One-Step qRT-PCR Kit is tested on a lot-to-lot basis to ensure consistent product quality.

Required Materials

  • Reaction tubes and caps /PCR plates and seals
  • qPCR thermal cycling instrument
  • DEPC- treat water
  • Gene-specific primers and fluorogenic probe
  • ROX reference dye (optional)
  • RNase Inhibitor (optional)

One-Step qRT-PCR Kit Protocol

  1. Thaw the One-Step qRT-PCR Kit, template RNA, primers, fluorogenic probe, ROX reference dye (optional) and DEPC-treat water on ice. Mix each solution well.

Set up the following reaction mixture, and reaction cocktails can be made when multiple reactions are being assembled.

Component Volume (μl) Final Concentration
2X Reaction Mix 12.5 µl 1X
Template RNA Variable 1 pg-1 µg
Sense Primer (10 µM) 0.5 µl 200 nM
Anti-Sense Primer (10 µM) 0.5 µl 200 nM
50 mM MgSO4 (optional) 0.5 µl
RT/ HotStar Taq Mix 0.5 µl
Fluorogenic probe (10 μM) 0.25 μl 100 nM
ROX Reference Dye (optional) Variable
RNase Inhibitor (optional) 0.5 µl
Add DEPC treat water to 25 µl

Note:

  1. Gently mix and make sure that all the components are at the bottom of the amplification tube. Centrifuge briefly if needed.
  2. Perform qRT-PCR reactions using the following cycling program:

Standard Mode Process:

Process Temperature (°C) Time Cycles
cDNA synthesis 50 15 minutes 1
RTase Inactivation / Hot-start Taq Activation 95 2 minutes 1
Denaturation 95 15 seconds 40
Annealing 60 30 seconds

Fast Mode Process:

Before set-up, choose the fast mode on the qPCR thermal cycling instruments

Process Temperature (°C) Time Cycles
cDNA synthesis 50 5 minutes 1
RTase Inactivation / Hot-start Taq Activation 95 2 minutes 1
Denaturation 95 3 seconds 40
Annealing 60 30 seconds

 

  1. Collect the data and analyze results.

Troubleshooting

Refer to the table below to troubleshoot problems that you may encounter when you did qRT-PCR amplification with the kit.

Problem Cause Solution
No signal, or one or Wrong cycling conditions Always start with the optimized cycling conditions
more signals detected specified in the protocols.
late in PCR Be sure that the cycling conditions include the initial step
for cDNA synthesis, RTase Inactivation / Hot-start Taq
Activation, and the specified times for denaturation and
annealing/extension.
 

 

Problem Cause Solution
No signal, or one or Wrong or no detection step Ensure that fluorescence detection takes place during
more signals detected the combined annealing/extension step.
late in PCR
Problems with starting Check the concentration, storage conditions, and quality
template of the starting template. If necessary, make new serial
dilutions of template nucleic acid from the stock solutions.
Repeat the PCR using the new dilutions.

Related Ordering Information

Cat. No. Description Size
SN017-0100 Total RNA Isolation Kit (Blood/ Cultured Cell/ Fungus) 100 Reactions
SN020-0100 Total RNA Isolation Kit (Plant) 100 Reactions
SN021-0100 Total RNA Isolation Kit (Tissue) 100 Reactions
SR001-2500 RiboIN RNase Inhibitor 2500 U

Caution

  • During operation, always wear a lab coat, disposable gloves, and protective equipment. 
  • Research Use Only. Not intended for any animal or human therapeutic or diagnostic uses