Mastermix 16S Complete Kit
Mastermix 16S Complete is a DNA-free 2.5x master mix with universal 16S rDNA primers, for the detection and identification of bacteria by PCR. The amplicon is about 450 bp and contains variable sequences, so the bacterium can be identified by taxon-specific probing or by sequencing. Research use only. The DNA-free grade is the point of this product: the manufacturer specifies an absence of signal in negative controls in at least 97 per cent of cases over up to 40 cycles.
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This kit in detail
- Universal 16S rDNA primers, amplicon of about 450 bp
- DNA-free grade: clean negative controls in at least 97 per cent of cases at 40 cycles
- 2.5x master mix with MolTaq 16S polymerase, DNA-free water and gel reagents
- Detection by gel electrophoresis, real-time PCR or array
- No internal control; positive control DNA not supplied
- 100, 250 or 1000 reactions per kit, REF S-020-0100, S-020-0250 and S-020-1000
Mastermix 16S Complete amplifies a region of the bacterial 16S rRNA gene with universal primers, for the detection and identification of bacteria in a DNA extract.
Why DNA-free matters
A universal 16S PCR amplifies any bacterial DNA, including the traces present in ordinary reagents and enzymes. That background sets the detection limit of the assay. This master mix is manufactured DNA-free, and the specification is that negative controls stay clean in at least 97 per cent of cases for up to 40 cycles.
Method
The amplicon of about 450 bp can be read on gel, by real-time PCR or on an array, and its variable sequences allow identification by taxon-specific probes or by sequence analysis. The reaction volume is 25 µl in the standard protocol and 20 µl in the LightCycler protocol. Cycling runs 1 minute at 95 °C followed by 40 cycles of 5 seconds at 95 °C, 5 seconds at 55 °C and 25 seconds at 72 °C. The protocol is validated on the Eppendorf Mastercycler Gradient, with real-time variants for the Bio-Rad Opticon, the Applied Biosystems StepOne and the Roche LightCycler 1.5 and 2.0; other cyclers have to be validated by the user.
What is not supplied
There is no internal control. DNA-free water serves as the negative control. For the positive control, genomic DNA from Staphylococcus aureus or another species is used at 0.5 to 5 ng per reaction, or the separate Molzym DNA positive control (S-200-050). Sequencing primers SeqGP16S and SeqGN16S are sold separately (S-775-100). The master mix is stable for 12 months and primer dimers can appear, including in the negative control.
Regulatory status
This kit is for research use only and not for use in diagnostic procedures.
The Molzym Mastermix 16S Complete is a PCR reagent that is supplied as a 2.5x-concentrated solution, containing all the necessary components for bacterial DNA amplification except for the template and DNA-free water. The following components are included in the kit:
- MolTaq 16S: This is a DNA polymerase that is optimized for bacterial DNA amplification.
- Buffer: The buffer contains salts and other components necessary for the PCR reaction to occur, such as magnesium ions.
- dNTPs: These are deoxynucleotide triphosphates, which are the building blocks for DNA synthesis.
- Validated primers: These primers are designed to target conserved regions of the 16S rRNA gene and are optimized for bacterial DNA amplification.
- SYBR Green 1 DNA staining solution: This solution is used for visualizing the amplified DNA in gel electrophoresis.
- DNA marker: This is a ladder of DNA fragments of known sizes that is used to estimate the size of the amplified DNA in gel electrophoresis.
- Gel loading solution: This is a loading buffer that is used to prepare the PCR products for gel electrophoresis.
The kit does not include the template DNA or DNA-free water, which need to be added by the user. Additionally, the kit includes a comprehensive user manual that provides detailed instructions for the preparation and use of the Molzym Mastermix 16S Complete, as well as troubleshooting tips and advice on data interpretation.
Datasheets and regulatory documentation
Everything your quality team needs for evaluation and procurement.
Instructions for Use
Frequently paired with this kit
Other assays and instruments lab teams add alongside the Mastermix 16S Complete Kit.
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