The NGS Cell Free DNA Library Prep Kit (illumina and MGI Platforms) was developed for the construction of high quality cell-free DNA (cfDNA) libraries using 1 ng to 50 ng of cell-free DNA as input. The kit has a simple work flow and a fast procedure. Multiplexing of the cell free DNA library is possible based on the index type.

NGS Cell Free DNA Library Prep Kit Workflow

The main source of cell-free DNA (cfDNA) is derived from apoptotic hematopoietic cells in blood and found in the plasma. The length of the cfDNA is about 150-200 bp in length. Circulating tumor DNA (ctDNA) derived from malignant tumors is a part of cfDNA. Both cfDNA and ctDNA can be used as a noninvasive biomarker since it offers a better approach in comparison to invasive tissue biopsies.

NGS has been used for cfDNA and ctDNA sequencing in the field of liquid biopsy as it provides a whole genome level of molecular profiling. One of the hurdles for cfDNA sequencing is the difficulty of library preparation from the limited amount of cfDNA obtained from plasma. Our cell-free NGS kit makes it easy to get enough libraries from limited input in just 1.5 hours.

Three index types are available for the NGS Cell Free DNA Library Prep Kit of the illumina platform:

Non-index (Cat.# 30029): Libraries do not have index.

Index(Cat.# 30031): Each of our index primers contains a unique 6-base index sequence that can be used for sample identification. Total 48  library multiplexing is possible. Index information can be downloaded here.

Unique dual index (Cat.# 30033): Cell-free DNA library multiplexing up to 96 samples is possible with the unique dual indexes. We have developed a Four-Base Difference Index System. The system have at least 4 bases different from each other in the 8 bases index length. The primers effectively minimize sequencing errors such as mis-assignment, index hopping, index contamination etc. Index information can be downloaded here.

Indexes are available for the MGI platform kits (Cat.# 34031).

Kit advantages:

• Fast and simple protocol
  • Hands-on time is around 10 minutes
  • The total protocol time is only 1.5 hours
• Easy procedure based on:
  • Ready-to-use master mix for easy setup of reactions
  • Less reaction components to simplify bench work
• Less magnetic beads used for cleanup procedures: This can reduce more than 50% of the beads consumption
• Guaranteed high library conversion efficiency
• Low input cell-free DNA: From 1 ng to 50 ng

Comparison of library conversion efficiency with different samples under the same condition.

Comparison of library yield with different samples under the same condition.

The NGS Cell Free DNA Library Prep Kit (illumina and MGI Platforms) was developed for the construction of high quality cell-free DNA (cfDNA) libraries using 1 ng to 50 ng of cell-free DNA as input. The kit has a simple work flow and a fast procedure. Multiplexing of the cell free DNA library is possible based on the index type.

Introduction

This product is suitable for rapid extraction of total DNA from tissue, cells, blood, saliva, swabs, blood spots, semen and other clinical samples. DNA can be used directly for PCR, quantitative PCR, Southern Blot, test of virus DNA and so on.

Details

Specifications

Features	Specifications
Main Functions	Isolation total DNA from tissue / blood / body fluid / swab /dry blood spots
Applications	PCR, qPCR, southern bolt and virus detection, etc
Purification method	Mini spin column
Purification technology	Silica technology
Process method	Manual (centrifugation or vacuum)
Sample type	Tissue, cell, blood, saliva, swab, blood spot,semen and other clinical samples
Sample amount	Solid tissue: 1-10mg; Anticoagulant blood: 200μl
Elution volume	≥20μl
Time per run	30 – 60 minutes
Liquid carrying volume per column	800µl
Binding yield of column	100µg

Principle

This product is based on silica Column purification. The sample is lysed and digested with lysate and protease, DNA is released into the lysate. Transfer to an adsorption column. Nucleic acid is adsorbed on the membrane, while protein is not adsorbed and is removed with filtration. After washing proteins and other impurities, Nucleic acid was finally eluted with low-salt buffer (10mm Tris, pH9.0, 0.5mm EDTA).

Advantages

• High quality DNA – meet a variety of downstream applications, including PCR, qPCR, enzyme digestion, hybridization, etc.
• Fast – without separation of leukocytes, organic extraction or ethanol precipitation
• Simple – all nucleic acids can be obtained by direct digestion
• Wide applicability- handle a variety of liquid samples

Kit Contents

Contents	D301802	D301803
Purification Times	50	250
HiPure DNA Mini Columns I	50	250
2ml Collection Tubes	100	500
Buffer ATL	30 ml	150 ml
Buffer AL	30 ml	150 ml
Buffer GW1	22 ml	88 ml
Buffer GW2	12 ml	50 ml
Proteinase K	24 mg	120 mg
Protease Dissolve Buffer	1.8 ml	10 ml
Buffer AE	15 ml	60 ml

Storage and Stability

Proteinase K should be stored at 2-8°C upon arrival. However, short-term storage (up to 12 weeks) 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. 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 when used.

Experiment Data

This product is suitable for rapid extraction of total DNA from tissue, cells, blood, saliva, swabs, blood spots, semen and other clinical samples. DNA can be used directly for PCR, quantitative PCR, Southern Blot, test of virus DNA and so on.

Overview

• Robust Lysis Solution processes even the most challenging plant species such as pine needle and grape 
• No phenol extractions
• DNA and all sizes of RNA are recovered, including microRNA
• High quality DNA and RNA are purified simultaneously using the same spin column
• No need to split the lysate​
• Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix

This kit provides for rapid spin column isolation and purification of total RNA and genomic DNA simultaneously from a single plant sample without splitting the lysate. Norgen’s plant lysis solution is highly robust and effective over a wide range of plants including challenging samples.  The total RNA and genomic DNA are both column purified in under 30 minutes.   Since RNA and DNA are isolated without splitting the lysate, variability and inconsistent results are reduced.​​ All sizes of RNA including microRNA are recovered without the need for phenol.  Optional on-column DNase and RNase treatments provide flexibility to isolate DNA-free RNA or RNA-free DNA respectively.  Isolated nucleic acids are of a high quality and yield, and are ready for downstream use including PCR, qPCR, RT-PCR, qRT-PCR, sequencing and more. 

Background

It is often necessary to isolate total RNA and genomic DNA from a single plant sample, such as for studies of gene expression, mutant or transgenic plant characterization, and host plant-pathogen characterization. This is of great benefit when isolating RNA and DNA from precious, difficult to obtain or very small samples. Furthermore, gene expression analysis will be more reliable since the RNA and DNA are derived from the same sample, therefore eliminating inconsistent results.

Details

Supporting Data

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Kit Specifications
Column Binding Capacity	50 μg for RNA 15 μg for genomic DNA
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	100 mg 5 x 106
Time to Complete 10 Purifications	30 minutes
Average Yields* Peach Leaves (100 mg)	40 μg RNA, 5 μg gDNA

* 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. These reagents should remain stable for at least 2 years in their unopened containers.

Component	Cat. 24400 (50 preps)
Lysis Buffer M	40 mL
Binding Buffer I	7 mL
Solution WN	18 mL
Wash Solution A	38 mL
Elution Buffer E	20 mL
Enzyme Incubation Buffer B	6 mL
Filter Columns	50
Spin Columns	50
Collection Tubes	100
Elution Tubes (1.7 mL)	50
Product Insert	1