Purify sequencing extension products from dye terminators, primers and other contaminants
Also purify DNA from different enzymatic reactions including restriction enzyme digests, Klenow reactions, alkaline phosphatase reactions, and ligations.
High recovery
Fast and efficient spin column format
Also available in 96 well format for high throughput
This kit provides a rapid spin column procedure for the purification and clean-up of sequencing and various other enzymatic reactions including restriction enzyme digests, Klenow reactions, alkaline phosphatase reactions, and ligations. The kit is used to remove reaction contaminants including dye terminators, salts, enzymes, excess primers and primer dimers. Contaminants are undesirable as they can interfere with many downstream applications including sequencing, RFLP, restriction enzyme digestions and ligation. Purification is based on spin-column chromatography without the use of phenol, chloroform or alcohol precipitation. The kit provides a high quality product with up to 90% recovery.
The kit is also available in a 96-well format for high-throughput sequencing reaction clean-up. Purification with the 96-well plate can be performed using either a vacuum manifold or centrifugation.
Storage Conditions and Product Stability All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 1 year after the date of shipment.
Component
Cat. 34500 (50 preps)
Cat. 34400 (2 x 96-well plate)
Binding Buffer C
30 mL
3 x 30 mL
Wash Solution A
12 mL
2 x 20 mL
Elution Buffer B
8 mL
2 x 15 mL
Spin Columns
50
–
Collection Tubes
50
–
Elution Tubes (1.7 mL)
50
–
96-Well Plate
–
2
Adhesive Tape
–
4
96-Well Collection Plate
–
2
96-Well Elution Plate
–
2
Product Insert
1
1
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96-Well 0.2 mL PCR Strip Rack SBS/ SLAS Footprint
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The Permagen 96-well 0.2 mL PCR Strip Rack was designed for use in manual or automation applications where strip tubes are required. It can also be used with our MSP750 MSP750RMSPU650R or MSPU650 magnet plates and is available as a discounted kit Picture shown is SKU: MSPU650RKT for reference
The Permagen 96-well 0.2 mL PCR Strip Rack was designed for use in manual or automation applications where strip tubes are required. It can also be used with
Propargyl-PEG2-amine is a crosslinker that is reactive with NHS esters, carbonyls (carboxylic acid, ketone, aldehyde). The propargyl group can participate in copper catalyzed azide-alkyne Click Chemistry reaction to form a stable triazole linkage. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.
Document
Propargyl-PEG2-amine is a crosslinker that is reactive with NHS esters, carbonyls (carboxylic acid, ketone, aldehyde). The propargyl group can participate in copper catalyzed azide-alkyne Click Chemistry reaction to form a stable triazole linkage. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.
Methylation Specific Bisulfite Seq Library Prep Kit
Product Info
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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.
