The Bisulfite Sequencing Library Prep Kit (illumina platform) was developed for construction of NGS libraries using bisulfite treated DNA (50 ng – 500 ng) as input. DNA methylation is an epigenetic mechanism known to play a critical role in gene regulation and genomic imprinting by blocking transcription factor access to promoters and enhancers. Bisulfite sequencing is a popular technique in biomedical research based on C to T conversion under the treatment of sodium bisulfite.

Recently, NGS became a powerful tool to identify the DNA methylation status at the whole genome level with single-base resolution. However, it is well known that bisulfite treatment of the NGS libraries causes tremendous damage to the libraries.

Bisulfite-Seq kit comparison

In the case of the regular bisulfite seq library preparation (library prep before bisulfite conversion), the DNA shearing equipment and the expensive methylated adaptors are required. In addition, the subsequent bisulfite conversion causes tremendous DNA damage to the constructed libraries.

BioDynami has developed a unique library prep technology to solve the problems. The technology uses bisulfite treated DNA as input to avoid the significant library loss caused by bisulfite conversion. Furthermore, DNA shearing step and expensive methylated adaptors are not required with our kit. The DNA polymerase in the kit has high-fidelity amplification ability and uracil tolerance which is ideal for amplification of bisulfite sequencing libraries. The final library is strand specific.

Bisulfite Sequencing Library Prep Kit Workflow

Three index types are available for the kit:

Non-index (Cat.# 30091): Libraries do not have index.

Index (Cat.# 30092): 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.# 30093): 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.

Kit advantages

• • • Easy and Quick protocol
      • Easy: Hands-on time only 10 minutes
      • Quick: Total protocol time around 1.5 hours
    • Simple workflow: Less steps
    • Directional library
    • Save magnetic beads more than 50%
    • Guaranteed high Bisulfite sequencing library conversion efficiency
    • Bisulfite treated DNA as input: From 50 ng to 500 ng

The Bisulfite Sequencing Library Prep Kit (illumina platform) was developed for construction of NGS libraries using bisulfite treated DNA (50 ng – 500 ng) as input. DNA methylation is an epigenetic mechanism known to play a critical role in gene regulation and genomic imprinting by blocking transcription factor access to promoters and enhancers. Bisulfite sequencing is a popular technique in biomedical research based on C to T conversion under the treatment of sodium bisulfite.

Overview

• Use with Norgen’s TaqMan Probe/Primer and Control Sets
• For any PCR application where the input template quality is important to avoid false negatives
• Convenience and time savings
• Cost efficient
• High sensitivity

TaqMan 2x PCR Master Mix – Cat. 28340

Norgen’s TaqMan 2x PCR Master Mix is a ready-to-use TaqMan 2x PCR Master Mix solution that contains a PCR internal control which can be detected by HEX/VIC channel in a real-time PCR machine. By detecting the internal control users can validate the DNA template quality, thereby preventing any false negatives in the PCR results. The user needs only to add template, target TaqMan primer/probe mix and water to set up the TaqMan real-time PCR. 

PCR Control: 
TaqMan 2x PCR Master Mix contains PCR control primers/probe (HEX/VIC) and PCR control template. The PCR control reaction in the TaqMan 2x PCR Master Mix is optimized to not interfere with target amplification. The fluorescence of the target probe should not be HEX/VIC.

Details

Reagents Supplied – Cat # 28340

• TaqMan 2x PCR Master Mix (3 Vials, 100 Reactions) – Sufficient reagent for 100 x 20 µL reactions

Storage Conditions and Product Stability
Norgen’s TaqMan 2x PCR and 2x RT-PCR Master Mixes should be stored at -20ºC. For everyday use an aliquot can be stored at 4ºC for up to three months. The Master Mix is stable for multiple freeze-thaw cycles. When stored at the proper temperature this reagent is stable for at least 1 year.

Introduction

The MagPure Plasmid purification system uses the paramagnetic bead technology for high-throughput preparation of high-copy or low-copy plasmid DNA from E. coli cells. This kit also can be used with fosmid and BAC vector-based constructs. The system uses alkaline lysis followed by a MagPure purification to differentially bind plasmid DNA to paramagnetic beads. While the DNA is bound to the beads, contaminants can be rinsed away using a simple washing procedure. Because MagPure uses magnetic separation technology, the protocol does not require vacuum filtration. This makes kit extremely amenable to automation. Plasmid DNA purified with this system is most commonly used in Sanger Sequencing and PCR amplification.

Details

Specifications

Features	Specifications
Main Functions	Isolation up to 15μg endotoxin free plasmid DNA from 1-5ml bacterial culture
Applications	Enzyme digestion, sequencing, PCR and labeling,  etc.
Purification technology	Magnetic beads technology
Process method	Manual or automatic
Sample type	Conventional plasmid, plasmid≤30KB
Sample amount	1-5ml
Elution volume	≥50μl
Time per run	≤80 minutes

Principle

This product is based on the purification method of high binding magnetic particles. The sample is lysed and digested under the action of lysate and Lysozyme. DNA is released into the lysate. After adding magnetic particles and binding solution, DNA will be adsorbed on the surface of magnetic particles, and impurities such as proteins will be removed without adsorption.The adsorbed particles were washed with washing solution to remove proteins and impurities, washed with ethanol to remove salts, and finally DNA was eluted by Elution Buffer.

Advantages

• High purity – purified plasmid can be directly used in sequencing, enzyme digestion and PCR, etc.
• Fast – it takes only 80 minutes to complete the isolation
• High yield – up to 15μg plasmid can be binded in one column
• Economic – excellent cost effectiveness performance

Kit Contents

Contents	P181402	P181403	P181404
Purification Times	100 Preps	500 Preps	5000 Preps
RNase A	10 mg	30 mg	2 x 160 mg
Buffer P1	30 ml	150 ml	2 x 800 ml
Buffer P2	30 ml	150 ml	2 x 800 ml
Buffer LEN3	20 ml	80 ml	800 ml
Buffer LN4	90 ml	400 ml	4 x 980 ml
MagPure Particles	3.5 ml	17 ml	3 x 60 ml

Storage and Stability

RNase A and MagPure Particles should be stored at 2–8°C upon arrival. However, short-termstorage (up to 12 weeks) at room temperature (15–25°C) does not affect its performance. Theremaining kit components can be stored dry at room temperature (15–25°C) and are stable for atleast 18 months under these conditions.The entire kit can be stored at 2–8°C, but in this case buffers should be redissolved before use. Make sure that all buffers are at room temperature whenused. If any precipitates form in the buffers,warm at 37℃ to dissolve. After addition of RNase A, Buffer P1 is stable for 6 months when stored at

The MagPure Plasmid purification system uses the paramagnetic bead technology for high-throughput preparation of high-copy or low-copy plasmid DNA from E. coli cells. This kit also can be used with fosmid and BAC vector-based constructs. The system uses alkaline lysis followed by a MagPure purification to differentially bind plasmid DNA to paramagnetic beads. While the DNA is bound to the beads, contaminants can be rinsed away using a simple washing procedure. Because MagPure uses magnetic separation technology, the protocol does not require vacuum filtration. This makes kit extremely amenable to automation. Plasmid DNA purified with this system is most commonly used in Sanger Sequencing and PCR amplification.