Introduction

HiPure Insect DNA Kits provides a simple and rapid solution for total DNA extraction of insect tissue samples. This kit is based on silica gel column purification technology without toxic phenol chloroform extraction and time-consuming alcohol precipitation. The whole extraction process only takes 30 minutes. HiPure Insect DNA Kit can process tissue samples less than 10mg at a time. Hipure Insect DNA 96 kit can process 96 insect tissue samples at a high throughput. The obtained DNA can be directly used in PCR, Southern blot, viral DNA detection and other experiments.

Details

Specifications

Features	Specifications
Main Functions	Isolation total DNA from <10 mg insect tissue
Applications	PCR, southern bolt and virus detection, etc
Purification method	Midi spin column
Purification technology	Silica technology
Process method	Manual (centrifugation or vacuum)
Sample type	Insect tissue samples
Sample amount	<10 mg
Elution volume	≥15μl
Time per run	≤30 minutes
Liquid carrying volume per column	800μl
Binding yield of column	100μg

Principles

This product is based on silica column purification. The sample is lysed and digested with lysate and protease, DNA is released into the lysate. Transfer to an adsorption column. Nucleic acid is adsorbed on the membrane, while protein is not adsorbed and is removed with filtration. After washing proteins and other impurities, Nucleic acid was finally eluted with low-salt buffer (10mm Tris, pH9.0, 0.5mm EDTA).

Advantages

• High purity DNA – can be used in sensitive downstream applications such as multiplex and quantitative pcr
• Fast – several samples can be extracted in less than 30 minutes
• Good repeatability – suitable for extracting high-yield DNA from insect tissue samples
• Safe – without phenol chloroform extraction and alcohol precipitation

Kit Contents

Contents	D312902	D312903
Purification Times	50	250
HiPure DNA Mini Columns I	50	250
2ml Collection Tubes	50	250
Buffer ITL	30 ml	120 ml
Buffer IL*	30 ml	120 ml
Buffer GW1*	22 ml	110 ml
Buffer GW2*	20 ml	2 x 50 ml
Proteinase K	24 mg	120 mg
Protease Dissolve Buffer	1.8 ml	15 ml
Buffer AE	15 ml	60 ml

Storage and Stability

Proteinase K should be stored at 2-8°C upon arrival. However, short-term storage (up to 12 weeks) at room temperature (15-25°C) does not affect their performance. The remaining kit components can be stored at room temperature (15-25°C) and are stable for at least 18 months under these conditions. The entire kit can be stored at 2–8°C, but in thiscase buffers should be redissolved before use. Make sure that all buffers areat room temperature when used.

HiPure Insect DNA Kits provides a simple and rapid solution for total DNA extraction of insect tissue samples. This kit is based on silica gel column purification technology without toxic phenol chloroform extraction and time-consuming alcohol precipitation. The whole extraction process only takes 30 minutes. HiPure Insect DNA Kit can process tissue samples less than 10mg at a time. Hipure Insect DNA 96 kit can process 96 insect tissue samples at a high throughput. The obtained DNA can be directly used in PCR, Southern blot, viral DNA detection and other experiments.

Overview

• Quantified standard to be used as a positive control or PCR quantification standard
• Vigorously quantified using multiple methods
• Includes partial sequence of STX-1 subunit A, and STX-2 subunit A genes (please refer to product insert for more details)
• Compatible with E. coli O157:H7 TaqMan PCR Detection Kit and E. coli O157:H7 TaqMan PCR Detection Probe/Primer and Control Set

E. coli O157:H7 is a rod-shaped, gram negative bacterium. It is an enterohemorrhagic strain of the common E. coli bacterium and infection by the O157:H7 strain is commonly associated with hemorrhagic colitis. E. coli O157:H7 is recognized by its somatic (cell wall) antigen (O157) and its flagella antigen (H7). In addition, E. coli O157:H7 is known to produce Shiga-like toxins, which cause severe symptoms. While most patients can recover from the infection, up to 15% of the patients may develop hemolytic uremic syndrome, a type of kidney failure that could be fatal. Infection of E. coli O157:H7 usually results from consumption of poorly prepared food including undercooked meat (particularly ground beef), untreated water or raw unpasteurized milk.

Norgen’s E. coli O157:H7 Quantified Bacterial DNA Standards are cloned fragments of the two Shiga-like toxin regions purified using Norgen’s sample preparation technology. They include partial sequences of STX-1 subunit A gene and STX-2 subunit A gene. Please refer to Appendix A for sequence information. The purified DNA is quantified vigorously using multiple methods including spectrophotometry, gel densitometry and real-time PCR. It is intended to be used as positive controls or PCR quantification standards for E. coli O157:H7. This product is compatible with Norgen Biotek’s E. coli O157:H7 Taqman PCR Detection Kit (Cat. TM41350) and E. coli O157:H7 Taqman PCR Detection Probe/Primer and Control Set (Cat. TM41310).

Details

Supporting Data

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Volume Provided	250 μL (for STX1)250 μL (for STX2)
DNA Quantity	2 x 104 copies per μL

Storage Conditions
Upon receipt, store Norgen’s E. coli O157:H7 Quantified Bacterial DNA Standards at -20°C or lower. Avoid multiple freeze-thaw cycles. If needed, prepare smaller working aliquots and store at -20°C or lower.

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.