For isolation and cultivation of Listeria monocytogenes.
Principle:
Peptone provide carbon and nitrogen sources; yeast extract powder and starch provide carbon and nitrogen sources, vitamins and growth factors; sodium chloride maintains osmotic equilibrium; glucose carbon source; agar as medium coagulant.
Norgen’s 2X One-Step RT-PCR Master Mix is a ready-to-use solution that contains components required for RT-PCR amplification of RNA templates. The mix includes M-MuLV reverse transcriptase, Taq DNA polymerase, dNTPs, reaction buffer, MgCl2, KCl, and a PCR enhancer/stabilizer. The user needs only to add the template, the primer set and water to the Master Mix to set up the RT-PCR reaction. This convenient 2X One-Step RT-PCR Master Mix reduces the time required to set up PCR reactions and reduces the possibility of contamination, particularly when preparing large numbers of reactions. The optimized master mix allows for robust amplification of RNA templates with high yields of PCR products.
Taq DNA Polymerase is a highly thermostable DNA polymerase that possesses a 5´→ 3´ polymerase activity and a very low 5´→ 3´ exonuclease activity. The source of Taq included with Norgen’s 2X One-Step RT-PCR Master Mix is an E. coli strain with a cloned Taq DNA Polymerase gene from Thermus aquaticus YT-1. M-MuLV Reverse Transcriptase is an RNA-directed DNA polymerase that can synthesize a complementary DNA strand initiating from a primer using either RNA (cDNA synthesis) or single-stranded DNA as a template. The source of the Reverse Transcriptase included with Norgen’s 2X One-Step RT-PCR Master Mix is an E. coli strain with a cloned reverse transcriptase gene from M-MuLV.
Norgen’s 2X One-Step RT-PCR Master Mix is available in 3 convenient sizes:
Cat # 28113 – 100 reactions (sufficient for 100 reactions x 20 µL reaction volume) Cat # 28114 – 200 reactions (sufficient for 200 reactions x 20 µL reaction volume) Cat # 28115 – 500 reactions (sufficient for 500 reactions x 20 µL reaction volume)
2X One-Step RT-PCR Master Mix (2 Vials, 100 Reactions each) – Sufficient reagent for 200 x 20 µL reactions
Storage Conditions and Product Stability 2X One-Step RT-PCR Master Mix should be stored at -20°C. For everyday use an aliquot can be stored at 4°C for up to 3 months. Repeated freeze-thaw cycles are not recommended. When stored at the proper temperature this reagent is stable for at least 1 year.
Buffer system mainly for stabilizing protein/enzyme and performance
B buffer
0.15ml
1 Tube
Mainly activated systems such as magnesium ions
Positive control template
0.1ml
1 Tube
Mainly the positive plasmid template is used to test the effectiveness of the kit
Positive control primer mix
0.06ml
1 Tube
Mainly the primer combination of the positive control template
Reagent Guide Manua
16T/bags,48T/Box
3 bags
Reagent technology of protein/enzyme system: freeze-dried powder, freeze-dried microspheres
Principle overview
This kit is based on a room temperature and constant temperature nucleic acid rapid amplification technology: at room temperature and constant temperature, the recombinase and primer form the protein/single-stranded nucleotide complex Rec/ssDNA, with the help of auxiliary proteins and single-stranded binding protein SSB , invade the double-stranded DNA template; form a D-loop region at the invasion site, and start scanning the DNA double-strands; after finding the target region complementary to the primer, the Rec/ssDNA complex disintegrates, and the polymerase also binds to The 3′ end of the primer initiates chain extension. This kit relies on the action of exonuclease at 39 ºC, adding specific molecular probes designed based on the template, and using fluorescence monitoring equipment to achieve real-time monitoring of the amplification process of the target fragment.
Primer design
It is recommended to use primers with a length of 30-35 bp. Primers that are too short will affect amplification speed and detection sensitivity; primers are designed to avoid the formation of secondary structures that affect amplification; the amplicon length is recommended to be 150-300 bp, usually no more than 500 bp.
Fluorescent probe design
The probe sequence does not overlap with the specific primer recognition site, is 46-52 nt in length, and the sequence avoids palindromic sequences, internal secondary structures, and continuous repeated bases. The probe has four modification sites: the middle position ≥ 35 nt from the 5′ end is labeled with a dSpacer (tetrahydrofuran, THF) as the recognition site for exonuclease; the upstream of the THF site is labeled with a fluorescent group, and the downstream Label a quenching group, the distance between the two groups is 2-4 nt; THF is ≥15 nt from the 3′ end, and the 3′ end is labeled with a modifying group, such as an amine group, a phosphate group or a C3-Spacer.
Product features and advantages:
This kit has the advantages of high sensitivity, strong specificity,and short reaction time (only 20 minutes), and the reaction groups are in dry powder state, which is easy to operate and easy to store.
It can be applied to various brands of fluorescence quantitative PCR instruments, constant temperature fluorescence amplification instruments and other fluorescence detection equipment.
Document
This kit is based on a room temperature and constant temperature nucleic acid rapid amplification technology: at room temperature and constant temperature, the recombinase and primer form the protein/single-stranded nucleotide complex Rec/ssDNA, with the help of auxiliary proteins and single-stranded binding protein SSB , invade the double-stranded DNA template; form a D-loop region at the invasion site, and start scanning the DNA double-strands; after finding the target region complementary to the primer, the Rec/ssDNA complex disintegrates, and the polymerase also binds to The 3′ end of the primer initiates chain extension. This kit relies on the action of exonuclease at 39 ºC, adding specific molecular probes designed based on the template, and using fluorescence monitoring equipment to achieve real-time monitoring of the amplification process of the target fragment.
Solid Phase Adsorption Toxin Tracking (SPATT) is a biomimetic in-situ water monitoring tool that falls under an expanding umbrella of passive samplers. It serves to warn researchers of toxin-producing harmful algal bloom (HAB) developments early on. It has been popularized through its affordability, ease of use, and its ability to capture ephemeral events in marine, brackish, and freshwater environments. Its uptake of contaminants has been shown to be more similar than other sampling methods to that of aquatic species like bivalves, mussels, and clams. It provides an average bioavailable fraction of a toxin over deployment time that can be used to determine an overall toxin risk to organisms. The sampling period typically depends on the bioactivity at a site, ranging from 24 hours to 4 weeks in most cases.
A SPATT passively absorbs and desorbs extracellular compounds over its stretch of time at a sampling site; in an organism, a toxin would go through biochemical detoxification processes. Passive samplers have a higher sensitivity for more compounds and provide improved stability and preservation of these compounds within the resin. SPATT devices capture less commonly detected cyanotoxins (e.g. cylindrospermopsin) at lower concentrations than that of a grab sample (collected at one point in time). Grab samples are limited in scope and sensitivity, and underrepresent toxins like microcystin-LR, which is picked up very reliably through SPATT technology.
Uses HP20 that is widely applicable for many toxins.
Used to capture:
Cyanotoxin (e.g. microcystin and cylindrospermopsin)
Saxitoxin & derivatives (GNTXs, C-toxins), and other paralytic shellfish toxins (PSTs)
