[TP1000] ExcelTaq™ Taq DNA Polymerase, 5 U/μl, 500 U

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.

Description

The ExcelTaq™ Hot Start II DNA Polymerase is a mixture of an aptamer-based inhibitor and a recombinant thermo-stable Taq DNA polymerase designed for preventing or minimizing non-specific DNA amplification in PCR reaction. The inactivation of polymerase is achieved by a reversible binding of the aptamer to the polymerase at temperatures below 45°C. The aptamer inhibitor releases polymerase during normal PCR cycling. The aptamer-based inhibition omits the time-consuming initial activation step required by chemically modified or antibody-based hot start polymerases.

The high specificity and sensitivity of ExcelTaq™ Hot Start II DNA Polymerase allows sensitive detection from limited amount of DNA templates, such as 1 pg of cDNA or 1 fg of plasmid DNA. With a high DNA synthesis rate and high thermo-stability, the ExcelTaq™ Hot Start II DNA Polymerase allows reactions to be set up at room temperature and is suitable for common and specialized PCR applications

Features

• Aptamer-based hot start PCR
• Reversible enzyme inactivation
• Omits extra enzyme activation step
• Convenient for room temperature PCR set-up
• High yield and specificity of target amplicons
• Wide range of amplicon length (up to 10 kb)
• High sensitivity (as low as 1 fg of plasmid)

Applications 

• High specificity PCR
• Generation of PCR products for TA cloning
• Routine PCR, multiplex PCR, colony PCR, and RT-PCR

Storage

-20°C for 24 months 

The ExcelTaq™ Hot Start II DNA Polymerase is a mixture of an aptamer-based inhibitor and a recombinant thermo-stable Taq DNA polymerase designed for preventing or minimizing non-specific DNA amplification in PCR reaction. The inactivation of polymerase is achieved by a reversible binding of the aptamer to the polymerase at temperatures below 45°C. The aptamer inhibitor releases polymerase during normal PCR cycling. The aptamer-based inhibition omits the time-consuming initial activation step required by chemically modified or antibody-based hot start polymerases.

The high specificity and sensitivity of ExcelTaq™ Hot Start II DNA Polymerase allows sensitive detection from limited amount of DNA templates, such as 1 pg of cDNA or 1 fg of plasmid DNA. With a high DNA synthesis rate and high thermo-stability, the ExcelTaq™ Hot Start II DNA Polymerase allows reactions to be set up at room temperature and is suitable for common and specialized PCR applications

Overview

• Protocol optimized for DNA isolated from a diversity of samples including stool, soil, water, saliva, plant, urine, skin, and more
• Simple and quick workflow: library could be prepared in less than 5 hours
• Component of Norgen’s metagenomics workflow
• A single NGS run can be prepared with up to 384 unique dual-index libraries

Sample type purification kit guide 

The 16S V2-V3 Library Preparation Kit for Illumina consists of the reagents and components required for library preparation of the 16S V2-V3 amplicon libraries to be used for next-generation sequencing on Illumina platforms. All molecular reagents including primers, enzyme mixes, indexes, and buffers are provided. Instructions for PCR clean up with the AMPure XP Magnetic Beads (supplied by customer) are also included for rapid purification of nucleic acid products generated at two steps of the workflow. The library prep workflow could be used for purified DNA inputs from different sources including stool, soil, water, saliva, plant, urine, skin swab, vaginal swab, cheek swab, nasal swab, plasma/serum, tongue swab, gum swab, and others.

The 16S V2-V3 Library Preparation Kit for Illumina has a streamlined procedure that reduces the handling time such that the library prep procedure can be completed in approximately 4 hours (see diagram below). Input DNA is first subjected to targeted PCR to amplify the V2-V3 region of the DNA encoding 16S rRNA. The post-PCR reaction is then cleaned up using AMPure XP beads. Dual index primers are then added using a limited-cycle PCR. The indexed amplicons flanked by 5′ and 3′ barcoded adaptors are then cleaned using AMPure XP beads. The libraries are then ready for quantification, pooling and sequencing.

Details

Supporting Data

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Minimum amount of starting material:	2.5 µL of DNA (5 ng/µL)
Time to complete library preparation:	4 hours

Storage Conditions and Product Stability
Norgen’s 16S V2-V3 Library Prep Kit for Illumina is shipped as one kit box (for the 24 prep kit) or two sub-component kits (for the 96 prep kit). All kits should be stored at -20°C upon arrival.

All kit components should remain stable for at least 1 year when stored at the specified storage conditions.

Step	Component	Cat. 70300 (24 preps)	Cat. 70310, 70320, 70330, 70340 (96 preps)
Amplicon PCR (PCR 1)	MGX Master Mix	330 µL	1,320 µL
	16S V2-V3 Primer Mix	70 µL	280 µL
Index PCR (PCR 2)	Indexing Master Mix	660 µL	2 x 1,320 µL
	N7xx Index Primer	50 µL	50 µL
	S5xx Index Primer	70 µL	70 µL
PCR Clean-Up	Resuspension Buffer	2 x 1,250 µL	2 x 5,000 µL
	Nuclease-free water	1,250 µL	1 x 6,000 µL