NGS TPH DNA Library Prep Set for Illumina (50 ng)

Cat. No.: N665730

Overview

Products content

N665730	Component	24 T	96 T	Storage
N665730A	TPS V50	144 μL	576 μL	-20℃. Avoid freeze/thaw cycle.
N665730B	5×FA Reaction Buffer	144 μL	576 μL	-20℃. Avoid freeze/thaw cycle.
N665730C	2×HiFidelity PCR Mix	600 μL	2×1.2 mL	-20℃. Avoid freeze/thaw cycle.
N665730D	PPM	48 μL	192 μL	-20℃. Avoid freeze/thaw cycle.

* This kit is suitable for human genomic DNA library construction with a starting template DNA input of 50 ng. We also have transposase library construction kits for human genomic DNA starting at 5 ng and 1 ng, so it is recommended to use different kits for different starting amounts of DNA in order to obtain higher quality libraries.

Products Introduction

This kit is developed for Illumina's high-throughput sequencing platform and provides the enzyme premix system and reaction buffer for genomic DNA library construction, including all components except PCR primers. Compared with the traditional library construction kits, this kit adopts the new transposase method for library construction, which can complete DNA fragmentation, end repair and junction reaction in one simple enzymatic reaction, significantly reducing the amount of template, reducing the number of experimental steps, and shortening the time of library construction; it adopts the high-fidelity DNA polymerase for library enrichment, and the preference-free PCR amplification can expand the coverage area of the sequence, which can be used for efficient and effective sequencing. The use of high-fidelity DNA polymerase for library enrichment and preference-free PCR amplification broadens the coverage area of the sequence and enables efficient preparation of DNA libraries for Illumina's second-generation sequencing platform. The kit is suitable for DNA libraries with a starting template of 50 ng, and all reagents in the kit have been subjected to strict quality control and functional validation to maximize the stability and reproducibility of library construction.

Product Features

● DNA fragmentation and junction ligation in one step.

● Ultra-fidelity amplification minimizes amplification-preferred steps.

Provide your own instruments, kits and consumables

1. Magnetic frame: DynaMagTM-2 is recommended.

2. DNA purification and recovery kit: It is recommended to use  DNA purification and recovery kit by magnetic bead method.

3. Library PCR primer kits:  transposase method for second-generation sequencing multi-sample primer kits are recommended.

4. Anhydrous ethanol, deionized water (pH between 7.0 and 8.0).

5. Reaction tubes: It is recommended to use low adsorption PCR tubes and 1.5 ml centrifuge tubes. Tips: It is recommended to use high quality filter tips to prevent contamination of kits and library samples.

Pre-experiment Preparation and Important Notes

1. Avoid repeated freezing and thawing of reagents.

2. PCR products are easily contaminated due to improper operation, resulting in inaccurate results. It is recommended to isolate the PCR reaction system preparation area from the PCR product purification area, and to use special pipettes to clean the experimental areas at regular intervals.

3. Bead purification: the beads should be equilibrated to room temperature before use, all operations on the beads should be carried out at room temperature, 80% ethanol should be dispensed freshly, the beads should be rinsed and dried until the surface is free of liquid reflections and has a frosted appearance, insufficient drying of the beads will cause ethanol residue that will affect the subsequent experiments, and over-drying of the beads will affect the efficiency of DNA recovery.

4. The kit is suitable for human genomic DNA library construction, if the DNA sample is a PCR product, it should be ensured that its length>.

500 bp, since transposases do not work on DNA ends, it is recommended to extend the PCR product by 50-100 bp at each end of the PCR product to avoid low coverage of the ends for sequencing.

Sample Preparation

DNA purity requirements: A260/A280 = 1.8-2.0. Sample DNA: dissolve in ultrapure water. DNA Quantification: Too much or too little DNA will affect the quality of the library. It is recommended to use Nano to test the purity of the genomic DNA and then use Qubit to test the concentration of the genome (do not use any absorbance-based assay for template quantification).

Schematic diagram of DNA banking process

procedure

DNA fragmentation, junction reaction

1. Add the following reagents to a 200 μl PCR tube:

 

2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.

3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows:

DNA should be purified immediately after the fragmentation reaction has been performed and the transposase is still in a high state of activity.

to prevent smaller library fragments due to DNA over-fragmentation.

Purification of fragmentation products

We recommend the use of the  Century Magnetic Bead Method DNA Purification and Recovery Kit.

1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.

2. Add 50 μl of magnetic beads equilibrated to room temperature to the fragmentation product, vortex and shake for 5 seconds, then let stand at room temperature for 5 minutes.

3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (approximately 3-5 minutes), carefully aspirate the supernatant and discard, avoiding contact with the beads that have bound the target DNA. Note: Do not discard the beads.

4. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 μl of freshly prepared 80% ethanol to the centrifuge tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.

Note: When adding ethanol, the liquid must not be blown directly onto the beads.

5. Repeat step 4.

6. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, then add 23 μlddH2O to solubilize.

Note: Do not over-dry the beads as this may affect the elution efficiency.

7. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer 21 μl of supernatant to a new 200 μl PCR tube.

PCR amplification

  Add the following reagents to the 200 μl PCR tube:

 Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.

3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows:

Selective recovery of library DNA fragments

It is recommended to use CombiVision Magnetic Beads DNA Purification and Recovery Kit for selective recovery of DNA fragments. When different sizes of DNA fragments are required, the amount of magnetic beads to be used is different, please refer to the attached table for the specific amount of magnetic beads to be used (if other brands of magnetic beads are used, you need to find out the optimal amount of magnetic beads to be used on your own).

Note: Amplification products can also be fragment length sorted and purified using the Gum Recovery Kit. If there is no special requirement for library length distribution, the amplification products can also be purified without selective recovery of DNA fragments as described on page 6 of the manual.

1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.

2. Transfer the PCR product to a 1.5 ml centrifuge tube, rehydrate to 100 μl and add several volumes of magnetic beads equilibrated to room temperature, vortex for 5 seconds and let stand at room temperature for 5 minutes.

3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, and carefully aspirate the supernatant and transfer it to a new 1.5 ml centrifuge tube.

Note: Do not discard the top clear.

4. Add several volumes of magnetic beads to the supernatant, vortex and shake for 5 seconds, then let stand at room temperature for 5 minutes.

5. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA.

Note: Do not discard the beads.

6. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 μl of freshly prepared 80% ethanol to the tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.

Note: When adding ethanol, the liquid must not be blown directly onto the beads.

7. Repeat step 6 once.

8. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 20 μl of ddH2O to solubilize.

Note: Do not over-dry the beads as this may affect the elution efficiency.

9. Remove the centrifuge tube from the magnetic rack, vortex and oscillate to completely resuspend the beads, and let stand at room temperature for 5 minutes. Leave briefly

centrifuge, place the tube on a magnetic rack until the solution is clear, and transfer the supernatant solution to a new centrifuge tube.

Table: Suggested amount of magnetic beads for different segment selection recovery

Library DNA fragment purification

We recommend the use of the  Century Magnetic Bead Method DNA Purification and Recovery Kit.

1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.

2. 50 μl of magnetic beads equilibrated to room temperature were added to the PCR product, vortexed and shaken for 5 seconds, and then left to stand at room temperature for 5 minutes.

3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (approximately 3-5 minutes), carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA. Note: Do not discard the beads.

4. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 μl of freshly prepared 80% ethanol to the centrifuge tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.

Note: When adding ethanol, the liquid must not be blown directly onto the beads.

5. Repeat step 4.

6. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 25 μl of ddH2O to solubilize.

Note: Do not over-dry the beads as this may affect the elution efficiency.

7. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer the supernatant solution to a new tube.

Library quality control

Determination of library concentration

In order to obtain high-quality sequencing results, accurate quantification of DNA libraries is required, and the first recommendation is to use Real-time

PCR methods are used for absolute quantification of DNA libraries. Additionally, fluorescent dye methods such as the Qubit method or the fluorescent dye picogreen method can be used; do not use quantification methods based on absorbance measurements here. The following approximate formula can be used to convert the molar concentration of the DNA library.

Average total length of libraries	Approximate conversion formula

 

Library fragment distribution

The prepared DNA libraries can be detected by agarose gel electrophoresis or Agilent 2100 Bioanalyzer.

Range of segment length distributions.

 

Storage and Shipping

Storage

Store at -20°C,Avoid repeated freezing and thawing

Shipped In

Ice chest + Ice pads