If you are detecting bacterial sequences other than E.coli, the primers will have to exclude any part of the DNA that is shared between E.coli and the bacteria used (in other words, the primers have to be designed only in the part which is unique for the bacteria used).
If detecting E.coli sequences, There are some traces of bacterial DNA in the Taq polymerase and in the UNG (as it is produced in E.coli). You will have to determine the minimum level and subtract it from the positive signal. Everything above this signal should be considered as positive.
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When designing Molecular Beacons, pay attention to the folding of the probe. It might fold into alternate conformations, which are not well quenched. Change the stem or loop sequence, or both to avoid this.
If the salt concentration of the buffer is too low (below 1 mM MgCl2) the probe does also not fold correctly.
Choose Eurogentec's Molecular Beacons and we will help you with the design.
Design assistance
In a one step RT qPCR reaction the RT reaction and the qPCR reaction are done in one and the same tube. The buffer is a combination of a RT and a PCR buffer, in which both enzymes do work. The one step RT qPCR reaction is a closed tube assay, so contamination can be avoided. It saves pipeting steps and time and is easy in handling.
In a two step RT qPCR the RT reaction and the qPCR reaction are done in separate tubes. It gives a more flexible way of working in that the cDNA can be used for more than one qPCR reaction and can be archived, so that it eliminates the need to continually isolate RNA.
Further more it allows the use of oligo d(T) and ramdom nonamer primers in the RT step and specific primers in the PCR step. This will increase the specificity and sensitivity of the assay.
Eurogentec offers both one and two step RT qPCR kits.
When performing a RT it is possible to use three different primer types:
Oligo d(T) primers, which bind to the poly A tail of the RNA and then only transcribe RNA. This will avoid contamination with genomic DNA. As the poly A tail is located at the beginning of the gene it will also lead to more full transcripts.
Random nonamers, which bind anywhere in the genome and allow the reverse transcriptase to fill up the gaps, will leads to high yields.
Specific primers, which bind to the gene of interest, and will therefore give specifics products.
The combination of oligo d(T) primers and random nonamers will give the highest yields and the longest transcripts, whereas specific primers transcribe only specific RNA but reduce the yield.
With an One step RT qPCR kit it is only possible to add specific primers, as it should be avoided that oligo d(T) primers and random nonamers participate in the PCR reaction, giving many aspecific products. As a RT reaction is performed at 40-50°C, the primers can bind with mismatches to the RNA and therefore transcribe unwanted sequences, which then also will be amplified in to consecutive PCR, leading to aspecific PCR products. This disadvantage is inherent to the method.
In a Two step kit the oligo d(T) primers and the random nonamers are included in the kit and will give ride to cDNA. Then two specific primers have to be selected and this will amplify the exact sequence.
When performing an assay with UNG, a first step at 50°C during 2min and a second step at 95°C during 10min has to be added before performing a normal qPCR:
50 °C during 2min
95 °C during 10min
94 °C during 15s
55 ºC-65 ºC during 30s
72 ºC during 60s
repeat during 35 cycles
Hold at 50 °C for ever
The fist step is needed to activate the UNG enzyme, to allow it to degrade U containing ds DNA.
The second step at 95 °C will deactivate the UNG and will activate the HotGoldStar.
For long term storage the Takyon™ qPCR MasterMixes and Core Kits should be stored at -15 °C to -25 °C in a constant temperature freezer. When stored under these conditions the reagents are stable during 12 months (MasterMixes) or 24 months (Core Kits).
For short term storage the Takyon™ qPCR MasterMixes and Core Kits can be stored at 4 °C for 6 months.
Goto TSKT to know more.
The Takyon™ Dry MasterMix dTTP can be stored at ambient temperature (15-35°C) up to 18-months. This is valid for unopened bottles (inert gas inside for long term stability)
!!! Do not expose the dried reagent to light as ROX normalisation dye is light sensitive
Learn about PCR and qPCR plastics and plate seals, what to look for when deciding which to use, and the impact they can have on the quality of DNA amplification studies. Browse the FAQs on PCR plates and PCR tubes; find resources to support your PCR and qPCR efforts with a focus on PCR plastics.
Ask yourself these questions to help determine which type(s) of PCR plastics you should use.
View tips on tackling PCR/qPCR issues potentially related to PCR consumables.
FAQs about PCR plates and qPCR plates
What are PCR plates?
PCR (Polymerase Chain Reaction) plates are specialized microplates designed for use in molecular biology research. They typically contain an array of 96 or 384 wells made of polypropylene, where each well holds an individual PCR or other reaction involving nucleic acids.
What is the difference between PCR and qPCR plates?
PCR plates are primarily used for standard PCR reactions, which involve amplifying DNA through a series of heating and cooling cycles. On the other hand, qPCR plates are used for quantitative PCR, which measures the amount of DNA present in a sample in real time. qPCR plate wells are typically made of white or frosted polypropylene to reduce background fluorescence, which can interfere with accurate detection of the target nucleic acid.
What are the benefits of using PCR plates over tubes?
Processing of multiple samples in parallel is easier in a PCR plate compared to individual tubes, saving sample preparation time and helping reduce user fatigue caused by capping and de-capping PCR tubes. Additionally, plates are more compatible with automated liquid handling systems, enabling further increases in throughput of PCR and qPCR workflows.
Can PCR and qPCR plates be used for other applications?
PCR and qPCR plates are well suited for many DNA and RNA applications including DNA sequencing, genotyping, and gene expression analysis. The versatility of these plates helps make them an essential item for molecular biology laboratories.
Can PCR and qPCR plates be reused?
PCR and qPCR plates are recommended for single use only. Re-use of plates heightens the risk of cross-contamination and unreliable results. If you routinely use less than half a plate, consider options such as segmented PCR plates and PCR strip tubes.
Explore the features of Thermo Scientific PCR and qPCR plates
Search the FAQ database for more specific information about PCR plates
FAQs about PCR tubes and strips
What are PCR tubes?
PCR tubes are small, coned-shaped vessels made of polypropylene and used in Polymerase Chain Reaction (PCR) experiments. PCR tubes are designed to avoid evaporation of the PCR reaction mixture (comprising DNA templates, primers, nucleotides, and polymerase enzymes) and withstand repeated temperature fluctuations that occur during the PCR reaction.
What is a PCR strip?
PCR strips, aka PCR tube strips, typically consist of 8 or 12 PCR tubes joined together in a linear arrangement. A PCR strip may have attached caps or utilize a strip of caps for tube sealing.
Figure 1. PCR tubes and PCR strips. (
A) Individual PCR tube with domed cap. (
B) PCR strip tubes with 2 domed cap options: cap strip or individually attached caps.
What are PCR strips used for?
PCR strips provide a convenient and efficient format for conducting multiple PCR reactions in parallel, making them a popular choice in molecular biology laboratories. The use of PCR tube strips facilitates handling and organization of the samples as well as simultaneous processing of multiple samples, helping save time and reducing labor. PCR strips compatible with standard thermal cyclers can be easily placed into the instrument's block for thermal cycling.
Search the FAQ database for more specific information about PCR tubes
Explore the features of Thermo Scientific PCR tubes and strip tubes
For more PCR And qPCR Kitsinformation, please contact us. We will provide professional answers.
Spotlight articles—PCR plastics
Additional PCR technical resources
- PCR and qPCR plastics selection guide
- PCR and qPCR webinars
- User bulletin: How to Use MicroAmp Reaction Plates, Tube Strips, and Tubes
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