PEG3-(Amino-Tri-(Propargyl-PEG2-ethoxymethyl)-methane)-(Amino-Tri-(carboxyethoxymethyl)-methane)

• Screening of Cylindrospermopsin in water samples at or above 30ppt
• Format: 15 tests (5 samples at 2 dilutions/5 controls)
• 5 – 10X Sample Dilution Buffer
• 5 – 1X Sample Dilution Buffer
• 5- Negative Control
• 10- Fixed Volume Pipettes

Description

Attogene’ s Cylindrospermopsin Lateral Flow Kit can be used to detect Cylindrospermopsin in source water samples.

Format: Rapid-Water – Run Time: 30 Minutes, enough to run two samples (undiluted and a 10 fold dilution) and negative control.

Cylindrospermopsin (CYN) is a potent cyanotoxin synthesized by select species of cyanobacteria, prominently including Cylindrospermopsin raciborskii. It belongs to the tricyclic alkaloid class, exhibiting a molecular weight of approximately 415 Da. Structurally, cylindrospermopsin features an uracil ring fused with a hydantoin moiety, alongside a guanidino group, attributes that render it highly soluble and polar in aqueous environments.

Cylindrospermopsin is notorious for its profound toxicity towards aquatic organisms and its potential threat to human health through exposure via contaminated water and food sources. Consequently, rigorous monitoring protocols are essential in regions prone to cyanobacterial blooms, where cylindrospermopsin can accumulate in freshwater reservoirs and other aquatic habitats. In recognition of these risks, regulatory bodies such as the United States Environmental Protection Agency (EPA) have implemented an action level guideline. As of 2019, EPA 10-day drinking water health advisory for cylindrospermopsin recommended a threshold of 0.7 parts per billion (ppb), or 700 parts per trillion (ppt) for children under the age of six, and 3 parts per billion, 3000 parts per trillion for anyone older, to effectively manage cylindrospermopsin levels. This precautionary measure aims to uphold both environmental sustainability and public health integrity by minimizing exposure risks.  The EPA has also drafted a human health recreational water quality criterion to protect human health at 8,000ppt.

EPA 10-Day drinking water Health Advisories for Cylindrospermopsin: 

Do not Drink – 0.7 μg/L for bottle fed infants and preschool children, pregnant and nursing woman, elderly immunocompromised and liver conditions

Do not Drink – 3.0 μg/L for school age children to adults

Do Not Use – 20 μg/L

EPA Draft Human Health Recreational Ambient Water Quality Criteria to protect human health: 8 μg/L

Screening of Cylindrospermopsin in water samples at or above 30ppt

Format: 15 tests (5 samples at 2 dilutions/5 controls)

5 – 10X Sample Dilution Buffer

5 – 1X Sample Dilution Buffer

5- Negative Control

10- Fixed Volume Pipettes

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.

Methylation Specific Bisulfite Seq advantages

• • • Enrichment of methylated CpG sites
    • Single-base resolution
    • Low cost for sequencing
    • Fast
      • Total time: 1.5 hours
      • Hands-on time: 10 minutes
    • Simple workflow
    • Bisulfite treated DNA as input: From 20 ng to 500 ng

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.

Introduction

Usages:
For cultivating and enriching non-fastidious bacteria, and for evaluating the efficacy of disinfection.

Principle:
Peptone and beef extract powder provide nitrogen, vitamins, amino acids and carbon; sodium chloride to maintain osmotic equilibrium.

Formulation（per liter）:
Peptone 10.0g
Beef Extract 3.0g
Sodium Chloride 5.0g
Final pH 7.4 ± 0.2

How to use：
1.Suspend 18g in 1L of distilled water, stirring heated to boiling to completely dissolve, autoclave at 121℃ for 15 minutes.
2.Diluted and treated samples.

Quality control：
          　　

Item	The name and number of strain	Growth	Colony Color
1	Escherichia coli ATCC25922	Good	Turbid broth,slight precipitate
2	Staphylococcus aureus ATCC25922	Good	Turbid broth,slight precipitate

Storage: Keep container tightly closed, store in a cool, dry place, away from bright light. Storage period of 3 years.

Specifications: 250g/bottle

250g