Introduction

Free-circulating nucleic acids, such as tumor-specific extracellular DNA fragments and mRNAs in the blood or fetal nucleic acids in maternal blood, are present in serum or plasmausually as short fragments, <1000bp(DNA). HiPure Circulating DNA Midi Kit enables efficient purification of these circulating nucleic acids from human plasma, serum, or urine. The extracted products can be used for clinical in vitro detection.

Details

Specifications

Principle

This product is based on silica gel purification. The sample is lysed and digested with lysate and protease, DNA is released into the lysate. Transfer to an adsorption plate and filter 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 (10 Mm Tris,pH 8.0).

Advantages

• High yield – most optimal process, free DNA (>50bp) can be obtained to the maximum extent
• High concentration – low elution volume, ensuring high nucleic acid concentration
• High purity – low alcohol binding method, completely removing inhibitor and protein pollution
• High recovery – DNA can be recovered at thelevel of PG by silica gel column purification

Kit Contents

Storage and Stability

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

Free-circulating nucleic acids, such as tumor-specific extracellular DNA fragments and mRNAs in the blood or fetal nucleic acids in maternal blood, are present in serum or plasmausually as short fragments,

Description

FluoroDye™ DNA Fluorescent Loading Dye is a ready-to-use 6X DNA loading dye designed for fast qualitative electrophoresis analysis. Containing sensitive fluorescent dye with high specific affinity towards double stranded DNA (dsDNA), the FluoroDye™ Fluorescent DNA Loading Dye has negligible background and renders destaining process unnecessary. The FluoroDye™ DNA Fluorescent Loading Dye allows the user to immediately visualize electrophoresis result upon completion or to monitor the electrophoresis in real time. FluoroDye™ DNA Fluorescent Loading Dye is compatible with both the conventional UV gel-illuminating system as well as the less harmful long wavelength blue light illumination system. FluoroDye™ emission as bound to dsDNA is 522 nm, while its excitation peaks are at 270, 370 and 497 nm.

Features:

• Excellent for premix with DNA samples
• Sensitivity: 0.14 ng (DNA)
• A safer alternative to EtBr
• Compatibility: suitable to blue or UV light
• Increased cloning efficiency (blue light)

Composition 

FluoroDye™ DNA Fluorescent Loading Dye is stored in 6X concentration in 60% glycerol and buffered with Tris-HCl and EDTA, containing Bromophenol blue, Xylene cyanol FF and Orange G as tracking dyes.

Storage

Protected from light
 -20°C for 24 months

FluoroDye™ DNA Fluorescent Loading Dye is a ready-to-use 6X DNA loading dye designed for fast qualitative electrophoresis analysis. Containing sensitive fluorescent dye with high specific affinity towards double stranded DNA (dsDNA), the FluoroDye™ Fluorescent DNA Loading Dye has negligible background and renders destaining process unnecessary. The FluoroDye™ DNA Fluorescent Loading Dye allows the user to immediately visualize electrophoresis result upon completion or to monitor the electrophoresis in real time. FluoroDye™ DNA Fluorescent Loading Dye is compatible with both the conventional UV gel-illuminating system as well as the less harmful long wavelength blue light illumination system. FluoroDye™ emission as bound to dsDNA is 522 nm, while its excitation peaks are at 270, 370 and 497 nm.

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.