The kit offers the unique feature to isolate total RNA including small RNA and DNA from serum and plasma without the need to resort to the cumbersome phenol/chloroform extraction or a time consuming proteinase digest. RNA purified using the kit is ready for applications such as RT-PCR, Northern blotting, poly A+ RNA (mRNA) purification, nuclease protection, and in vitro translation.
Detail
Introduction
The kit offers the unique feature to isolate total RNA including small RNA and DNA from serum and plasma without the need to resort to the cumbersome phenol/chloroform extraction or a time consuming proteinase digest. RNA purified using the kit is ready for applications such as RT-PCR, Northern blotting, poly A+ RNA (mRNA) purification, nuclease protection, and in vitro translation.
Details
Specifications
Features
Specifications
Main Functions
Isolation total RNA and miRNA from 0.2-0.6ml serum and plasma
Applications
Purification method
Mini spin column
Purification technology
Silica technology
Process method
Manual (centrifugation or vacuum)
Sample type
Sample amount
Yield
Elution volume
Time per run
Liquid carrying volume per column
Binding yield of column
Principle
HiPure RNA technology simplifies total RNA isolation. The sample material is denatured in Lysis Buffer. The protein is then precipitated by Protein Precipitation Solution and pelleted by centrifugation. After the removal of protein the binding conditions for nucleic acids are adjusted by adding isopropanol. Total nucleic acids are bound to the column. Optionally, DNA can be removed by an on-column DNase digest. The remaining nucleic acids are washed and eluted with minimal amounts of RNase-Free water.
Kit Contents
Contents
R431402
R431403
Purification Times
50 Preps
250 Preps
HiPure RNA Mini Columns
100
2 x 250
2ml Collection Tubes
100
2 x 250
Buffer CFL
6 ml
30 ml
Buffer CFP
1.8 ml
10 ml
Buffer RWC
20 ml
80 ml
Buffer RW2*
20 ml
2 x 50 ml
RNase Free Water
10 ml
60 ml
Storage and Stability
The kit components can be stored at room temperature (15-25°C) and are stable for at least 18 months under these conditions.
Other Products
96 Well Plate for Enzyme Assays and Lateral Flow with Detachable Wells 10pack
Optically clear flat bottom plates with detachable wells allow for direct microscopic viewing and can be used with top and bottom reading instruments. Detachable wells give the user improved flexibility. Individual alphanumerical codes for easy identification. Minimum volume of 50uL maximum of 300.
Document
Set of 10 breakable well 96 well plates
Perfect for Lateral Flow or Enzyme Assays
Methylation Specific Bisulfite Seq Library Prep Kit
Product Info
Document
Product Info
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.
Document
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.
