Salmonella enterica have emerged as significant foodborne pathogens that pose a serious public health problem. The symptoms of salmonellosis may include diarrhea, fever, vomiting, and abdominal cramps with elderly, new-born, and immunocompromised individuals the most susceptible. S. enterica is a facultatively anaerobic Gram-negative bacterium that could survive low temperatures and freezing. The majority of the 1.3 billion annual cases of Salmonella-caused human gastroenteritis result from ingestion of contaminated food products, such as raw or undercooked meat, seafood, and eggs, as well as raw or unpasteurized milk and dairy products. Salmonella infections are also contracted following consumption of fresh fruits or vegetables that have been contaminated by infected fertilizer.
Salmonella enterica TaqMan PCR Kit, 100 reactions
Salmonella enterica TaqMan PCR Probe/Primer Set and Controls, 100 reactions
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Storage Conditions and Product Stability
All kit components can be stored for 2 years after the date of production without showing any reduction in performance.
All kit components should be stored at -20°C upon arrival.
| Component | Cat. TM32150 (100 preps) | Cat. TM32110 (100 preps) |
|---|---|---|
| MDx TaqMan 2X PCR Master Mix | 2 x 700 μL | – |
| S. enterica Primer & Probe Mix | 280 μL | 280 μL |
| S. enterica Positive Control | 150 μL | 150 μL |
| Nuclease-Free Water (Negative Control) | 1.25 mL | 1.25 mL |
| Product Insert | 1 | 1 |
This kit provides a fast, reliable and convenient method to isolate and concentrate exosomal RNA from previously purified exosomes. It allows for the isolation of RNA from intact extracellular vesicles (EVs) purified from different urine or plasma/serum sample volumes. The purification is based on Norgen’s proprietary resin.
The Exosomal RNA Isolation Kit is designed to isolate all sizes of RNA, including microRNA. Moreover, the kit allows the user to elute into a flexible elution volume ranging from 50 µL to 100 µL. The purified RNA is of the highest integrity, and can be used in a number of downstream applications including real time PCR, reverse transcription PCR, Northern blotting, RNase protection and primer extension, and expression array assays.
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| Kit Specifications | |
| Sample type | Exosomes purified using Norgen’s Purification Kits and most other methods |
| Size of RNA Purified | All sizes, including miRNA and small RNA (< 200 nt) |
| Elution Volume | 50-100 μL |
| Time to Complete 10 Purifications | 35-40 minutes |
| Average Yields* | Variable depending on specimen |
*Please check page 5 of the product insert for the average yields and the common RNA quantification methods.
Storage Conditions and Product Stability
All buffers should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment. It is recommended to warm Lysis Buffer A for 20 minutes at 60°C if any salt precipitation is observed.
| Component | Cat. 58000 (50 preps) |
|---|---|
| Lysis Buffer A | 20 mL |
| Lysis Additive B | 2 mL |
| Wash Solution A | 18 mL |
| Elution Solution A | 6 mL |
| Mini Spin Columns | 50 |
| Collection Tubes | 50 |
| Elution Tubes (1.7 mL) | 50 |
| Product Insert | 1 |
Bisulfite seq is a well know technology to detect DNA methylation and several technologies such as WGBS, RRBS, MeDIP-Seq, and MSBS are used for whole genome DNA methylation analysis. DNA methylation is important for regulation of cell development, differentiation and gene expression in molecular biology, genetics and epigenetics. Most methylated cytosines are found at CpG sites, and 70-80% of cytosines are methylated. The number of CpG sites in human genome is around 28 million, which is less than 1% of the genome compared with 4.4% expected.
Whole genome bisulfite sequencing (WGBS) is the most effective method of DNA methylation analysis. The only limitation is the sequencing cost is very high because the whole genome is sequenced including all the non-methylated regions.
Reduced Representation Bisulfite Sequencing (RRBS) is the reduced representation of a smaller fraction of the methylated CpG sites. RRBS combines restriction enzyme digestion and bisulfite sequencing, and enriches the sequencing for methylated CpG sites. It is an efficient technology for estimate the whole genome methylation patterns at the single base level. Although this allows a higher coverage depth and reduces the sequencing cost, the limitation is only 10% of the methylated CpG sites are covered.
Methylated DNA Immunoprecipitation Sequencing (MeDIP-Seq) is another whole genome enrichment technique used for selection of methylated DNA. Using antibodies against 5-methylcytosine, methylated DNA is enriched from whole genomic DNA via immunoprecipitation. 5-methylcytosine antibodies are incubated with fragmented genomic DNA and precipitated, followed by DNA purification and sequencing. There are several drawbacks of MeDIP-Seq: 1. Low resolution (150~200 bp) as opposed to the single base resolution; 2. Non-specific interaction due to antibody specificity and selectivity. 3. Bias towards hypermethylated regions.
The Methylation Specific Bisulfite Seq (MSBS) Library Prep Kit (illumina platform) was developed for construction of NGS libraries for methylated CpG sites using bisulfite treated DNA (20 ng – 500 ng) as input. The kit enriches methylated CpG regions, thus significantly reduce the sequencing cost. The kit estimates the whole genome methylation patterns at the single base level since it is based on a bisulfite-seq technology.
It is known that bisulfite treatment of completed NGS libraries causes tremendous damage to the libraries. By using bisulfite treated DNA as input, the kit overcomes the significant library loss due to the bisulfite conversion. The kit contains a mixture of PCR polymerases that have high-fidelity amplification and uracil tolerance which is ideal for bisulfite treated DNA.
Methylation Specific Bisulfite Seq Library Prep Kit Workflow
Three index types are available for the kit:
Non-index (Cat.# 30101): Libraries do not have index.
Index (Cat.# 30102): Each primer contains a unique barcode sequence of 6 bases to identify the individual library. Library multiplexing capacity is up to 48 samples. Index information can be downloaded here.
Unique dual index (Cat.# 30103): The multiplexing of bisulfite sequencing library is up to 96 samples with unique dual indexes. We used a Four-Base Difference Index System to generate indexes that have at least 4 bases different from each other in the 8-base index. The index primers remove NGS errors including index cross-contamination, index hopping, reads mis-assignment etc. Index information can be downloaded here.
MSBS Library Prep Kit enriches CpG sites
High methylation regions and low methylation regions in human genome.
High methylation region in human genome.
Low methylation region in human genome.
Sequencing setting: Single-end 35 cycles (Read 1, 35 bases) recommended
To maximize the methylated CpG enrichment, we recommend to sequence the MSBS libraries with single end 35 cycles (read1, 35 bases). This is because the enriched methylated CpG sites are mainly located around the beginning of read 1 sequences. Shorter single end reads tend to have better methylated CpG enrichment.
Bisulfite seq is a well know technology to detect DNA methylation and several technologies such as WGBS, RRBS, MeDIP-Seq, and MSBS are used for whole genome DNA methylation analysis. DNA methylation is important for regulation of cell development, differentiation and gene expression in molecular biology, genetics and epigenetics. Most methylated cytosines are found at CpG sites, and 70-80% of cytosines are methylated. The number of CpG sites in human genome is around 28 million, which is less than 1% of the genome compared with 4.4% expected.