Propargyl-PEG4-(CH2)3-methyl ester

Introduction

This product is suitable for extracting RNA  from 1ml anticoagulant blood (fresh or frozen blood/cryopreservation blood), lymphocytes, buffy coat, bone marrow, cultured cells, etc using Magzol reagents. It can specially extract high quality RNA from frozen blood samples.

Details

Specifications

Features	Specifications
Main Functions	Isolation total RNA from 1ml anticoagulant blood (fresh or frozen blood/cryopreservation blood), lymphocytes, buffy coat, bone marrow, cultured cells, etc using Magzol reagents.
Applications	RT-PCR, cDNA synthesis, second generation sequencing
Purification method	Polydisperse magnetic beads
Purification technology	Magnetic beads technology
Process method	Manual or automatic
Sample type	anticoagulant blood, lymphocytes, buffy coat, bone marrow, cultured cells
Sample amount	1ml whole blood (fresh or frozen blood)

Principles

This product is based on the purification method of high binding magnetic particles. The sample is lysed and digested by lysis buffer and protease, and RNA/DNA is released into the lysis buffer. Add binding solution and magnetic particles to adsorb RNA/DNA, while proteins are not adsorbed and removed. The particles adsorbed with DNA/RNA are washed with washing buffer to remove proteins and other impurities, then washed with ethanol to remove salt, and finally digested with DNase to remove DNA. RNA is recovered by adding binding solution, and finally the RNA is eluted with low salt buffer. The eluted RNA can be directly used for experiments such as RT-PCR, NGS and virus detection.

Kit Contents: Bottle

Contents	R661301	R661302	R661303
Purification Times	48 Preps	96 Preps	480 preps
DNase I	600 μl	2 x 600 μl	10 x 600 µl
DNase Buffer	20 ml	30 ml	150 ml
MagPure Particles N	2.5 ml	5.0 ml	28 ml
MagZol 3BD	65 ml	140 ml	3 x 200 ml
Buffer ALB2	40 ml	60 ml	350 ml
Buffer BCP2	10 ml	15 ml	80 ml
Buffer MW2*	20 ml	50 ml	2 x 100 ml
RNase Free Water	10 ml	20 ml	60 ml

Storage and Stability

DNase I should be shipped with ice pack or dry ice and stored at -20°C upon arrival. MagPure Particles N, MagZol 3BD and Buffer BCP2 should be stored at 2–8°C upon arrival. DNase I should be stored at -20°C. However, short-term storage 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.

This product is suitable for extracting RNA from 1ml anticoagulant blood (fresh or frozen blood/cryopreservation blood), lymphocytes, buffy coat, bone marrow, cultured cells, etc using Magzol reagents. It can specially extract high quality RNA from frozen blood samples.

Propargyl-PEG5-amine is a propargyl linker that is commonly used as a Click Chemistry reagent for copper-catalyzed reactions with azides. The amine moiety reacts with carboxylic acids, activated NHS esters and other carbonyl compounds. The PEG spacer helps improve the water solubility of the molecule in aqueous media. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.

Propargyl-PEG5-amine is a propargyl linker that is commonly used as a Click Chemistry reagent for copper-catalyzed reactions with azides. The amine moiety reacts with carboxylic acids, activated NHS esters and other carbonyl compounds. The PEG spacer helps improve the water solubility of the molecule in aqueous media. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.

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.