GeneAb™ CD15/Leu-M1

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.

Overview

• Extract total RNA, including virus & viroid RNA
• Robust lysis buffer is well-suited to even challenging samples such as pine needle, grape leaf, etc
• Isolate total RNA (including microRNA) without phenol
• Isolated RNA is of high quality, integrity and diversity
• Also available in 96-well format for high throughput applications
• Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix

Norgen’s Plant/Fungi Total RNA Purification Kit provides a rapid method for the isolation and purification of total RNA, including virus and viroid RNA, from a wide range of plants. Total RNA can be purified from fresh or frozen plant tissues, plant cells or filamentous fungi samples using this kit. All sizes of RNA are purified, including microRNA (miRNA) . The procedure is rapid and convenient. 

The RNA is purified without the use of phenol or chloroform. The purified RNA is of the highest quality, and can be used in a number of downstream applications including real time PCR, reverse transcription PCR, Northern blotting, RNase protection and primer extension, and expression array assays.  

Norgen’s Plant/Fungi Total RNA Purification Kit is also available in a 96-well (High Throughput) format for high throughput applications.  Purification with the 96-well plates can be performed using either a vacuum manifold or centrifugation.

Details

Supporting Data

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Kit Specifications – Spin Column
Maximum Column Binding Capacity	50 μg
Maximum Column Loading Volume	650 μL
Size of RNA Purified	All sizes, including small RNA (< 200 nt)
Maximum Amount of Starting Material: Plant Tissues Plant Cells Fungi	50 mg 1 x 106 cells 50 mg (wet weight)
Average Yield* 50 mg Tomato Leaves 50 mg Tobacco Leaves 50 mg Plum Leaves 50 mg Grape Leaves 50 mg Peach Leaves	60 μg 60 μg 32 μg 35 μg 30 μg
Time to Complete 10 Purifications	30 minutes

* Yield will vary depending on the type of sample processed.

* Yield will vary depending on the type of sample processed.

Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment.

Select Plants and Fungi Tested with the Norgen Plant/Fungi Total RNA Purification Kits

Plants

Tobacco (Nicotiana tabacum)
Tomato (Lycopersicon esculentum)
Pepper (Capsicum annuum)
Potato (Solanum tuberosum)
Arabidopsis thaliana1
Peach (Prunus persica)
Apple (Malus sp.)
Pear (Pyrus sp.)
Grape vine (Vitis sp.)
Plum (Prunus sp.)
Palm (Arecaceae)
Pine needle (Pinaceae)
Strawberry
Raspberry
Blackberry
Herbs
Persimmon (Ebenaceae)
Potato tuber (Solanum)
Plum fruit
Citrus
Vanilla bean
Cotton (Gossypium)
Mangrove
Chrysanthemum
Grape berry skin
Kiwi leaves
Peach (fruits and flowers)
Soy bean (legume)
Eastern White Red Cedar
Corn leaves
Cucumber leaves

Fungi

Aspergillus niger
Mucor racemosus
Cladosporium cladosporioides
Fusarium oxysporum
Penicillium sp.
Botrytis cinerea (Botryotinia fuckeliana)
Pichia sp.
Rhizopus oryzae
Alternaria tenuissima

Component	Cat. 25800 (50 preps)	Cat. 31350 (100 preps)	Cat. 25850 (250 preps)	Cat. 31900 (192 preps)
Lysis Buffer C	60 mL	1 x 30 mL, 1 x 60 mL	3 x 60 mL	2 x 60 mL
Wash Solution A	38 mL	38 mL	1 x 18 mL, 2 x 38 mL	2 x 38 mL
Elution Solution A	6 mL	6 mL	20 mL	20 mL
Filter Columns	50	100	250	–
Spin Columns	50	100	250	–
96-Well Plate	–	–	–	2
Adhesive Tape	–	–	–	4
Collection Tubes	100	200	500	–
96-Well Collection Plate	–	–	–	2
Elution Tubes (1.7 mL)	50	100	250	–
96-Well Elution Plate	–	–	–	2
Product Insert	1	1	1	1

Product Details

Kit Size:

48 Reactions

Product Name:

DNA Isothermal Rapid Amplification Kit Fluorescent Type

Kit Type:

DNA

Reaction Volume:

50 μL

Storage Temperature:

-20℃

Application:

Nucleic Acid Amplification

Type:

Fluorescent Type

Reaction Time:

20mins

High Light:

20mins home test kit

, 

DNA home test kit

, 

48 Reactions home dna test kit

Payment & Shipping Terms

Minimum Order Quantity

48T

Price

3.8$/T

Packaging Details

16T/bag,48T/box

Delivery Time

6days

Payment Terms

T/T,Paypal

Supply Ability

100000T/Month

Product Description

DNA Isothermal Rapid Amplification Kit(Fluorescent Type)Guide Manual

Product parameters:

Reagent component ( WLE8202KIT ,16T/bags,48T/Box )
Component	Specification	Quantity	Function
A buffer	1.6ml	1 Tube	Buffer system mainly for stabilizing protein/enzyme and performance
B buffer	0.15ml	1 Tube	Mainly activated systems such as magnesium ions
Positive control template	0.1ml	1 Tube	Mainly the positive plasmid template is used to test the effectiveness of the kit
Positive control primer mix	0.06ml	1 Tube	Mainly the primer combination of the positive control template
Reagent Guide Manua	16T/bags,48T/Box	3 bags	Reagent technology of protein/enzyme system: freeze-dried powder, freeze-dried microspheres

Principle overview

This kit is based on a room temperature and constant temperature nucleic acid rapid amplification technology: at room temperature and constant temperature, the recombinase and primer form the protein/single-stranded nucleotide complex Rec/ssDNA, with the help of auxiliary proteins and single-stranded binding protein SSB , invade the double-stranded DNA template; form a D-loop region at the invasion site, and start scanning the DNA double-strands; after finding the target region complementary to the primer, the Rec/ssDNA complex disintegrates, and the polymerase also binds to The 3′ end of the primer initiates chain extension. This kit relies on the action of exonuclease at 39 ºC, adding specific molecular probes designed based on the template, and using fluorescence monitoring equipment to achieve real-time monitoring of the amplification process of the target fragment. 

Primer design

It is recommended to use primers with a length of 30-35 bp. Primers that are too short will affect amplification speed and detection sensitivity; primers are designed to avoid the formation of secondary structures that affect amplification; the amplicon length is recommended to be 150-300 bp, usually no more than 500 bp. 

Fluorescent probe design

The probe sequence does not overlap with the specific primer recognition site, is 46-52 nt in length, and the sequence avoids palindromic sequences, internal secondary structures, and continuous repeated bases. The probe has four modification sites: the middle position ≥ 35 nt from the 5′ end is labeled with a dSpacer (tetrahydrofuran, THF) as the recognition site for exonuclease; the upstream of the THF site is labeled with a fluorescent group, and the downstream Label a quenching group, the distance between the two groups is 2-4 nt; THF is ≥15 nt from the 3′ end, and the 3′ end is labeled with a modifying group, such as an amine group, a phosphate group or a C3-Spacer. 

Product features and advantages:

This kit has the advantages of high sensitivity, strong specificity,and short reaction time (only 20 minutes), and the reaction groups are in dry powder state, which is easy to operate and easy to store.

It can be applied to various brands of fluorescence quantitative PCR instruments, constant temperature fluorescence amplification instruments and other fluorescence detection equipment.

This kit is based on a room temperature and constant temperature nucleic acid rapid amplification technology: at room temperature and constant temperature, the recombinase and primer form the protein/single-stranded nucleotide complex Rec/ssDNA, with the help of auxiliary proteins and single-stranded binding protein SSB , invade the double-stranded DNA template; form a D-loop region at the invasion site, and start scanning the DNA double-strands; after finding the target region complementary to the primer, the Rec/ssDNA complex disintegrates, and the polymerase also binds to The 3′ end of the primer initiates chain extension. This kit relies on the action of exonuclease at 39 ºC, adding specific molecular probes designed based on the template, and using fluorescence monitoring equipment to achieve real-time monitoring of the amplification process of the target fragment.