Fast Tagment DNA Library Prep Kit for Illumina (for 1ng) HYD121

Components

*Index sequence during sequencing: T501-CTCTCTAT (NovaSeq 6000 v1.0) or ATAGAGAG (NovaSeq 6000 v1.5), T701-TAAGGCGA, T702-CGTACTAG.

Storage Conditions

Store at -25~-15℃ for 1 year.

 Notes

About the operation

1. Please operate with lab coats and disposable gloves，for your safety.

2. Thaw components at room temperature. After thawing, mix thoroughly by vortexing, spin the tube briefly andplace them on ice for later use.

3. When preparing the reaction solution of each step, it is recommended to use a pipette to mix well or gently Vigorous shaking may cause a decrease in library output.

4. It is highly recommended to use filtered pipet tips to avoid cross-contamination. Be sure to change pipet tipswhen processing different samples.

5. Improper operations may very likely cause aerosol contaminations, impacting the accuracy of result. Mandatoryphysical isolation of PCR reaction mixing regions and PCR product purification assay regions is recommended. Equipped with equipment such as specialized pipettes for library preparation.Perform routine cleaning for each area by wiping the surfaces with 0.5% sodium hypochlorite or 10% bleach.

6. This product is for research use only.

About the product principle

This product is developed based on the transposase method, and its core principle is transposase and its transposition mechanism. The Transposome Mix contains a complete transposon composed of transposase and two equimolar adapters Adapter 1 and Adapter 2. When transposition occurs, the transposon inserts the adapter sequences of Adapter 1 and Adapter 2 into the target gene, forming a DNA with Adapter 1 at one end and Adapter 2 at the other end. Then, DNA polymerase is used to fill the incision formed by transposition. This product is amplified by two pairs of primers, N5 (N5XX) and N7 (N7XX) and P5 and P7 (PCR Primer Mix). After sorting and purification, the product becomes a sequenceable library.

DNA Fragmentation

1. This kit uses transposase for DNA fragmentation. If the DNA sample is a PCR product, its length should be >500 bp. Because transposase cannot act on the DNA ends, the sequencing coverage of the last 50 bp of the PCR product may be reduced. We recommend that you extend the two ends of the region to be tested by 50-100bp when preparing the PCR product to avoid the situation where the terminal sequencing coverage is reduced.

2. This kit is suitable for 1 ng Input DNA. Before the Input DNA is put in, it should be purified and dissolved in sterile distilled water, A260/A280 = 1.8-2.0, and quantified using double-stranded DNA fluorescent dye-based methods such as Qubit®, PicoGreen®, etc. Note: Because the Transposome Mix is very sensitive to DNA concentration, accurate DNA concentration determination is crucial to the success of the experiment.

Bead-based DNA Cleanup and Size Selection

1. The magnetic beads should be equilibrated at room temperature prior to use, otherwise the yield will decrease and the size selecting effect will be affected.

2. The magnetic beads should be mixed well by vortex or pipetting prior to use.

3. Do not aspirate the beads when transferring the supernatant, even trace amounts of the beads may impact the following reactions.

4. The 80% ethanol should be freshly prepared, otherwise it will affect the recovery efficiency.

5. For accurate size-selection, it is recommended to start with a volume of more than 100 μL. If less, it is recommended to bring the volume up to 100 μL with ultra-pure water.

6. The magnetic beads should be dried at room temperature before eluting the product. Insufficient dryness will easily cause ethanol residual to affect subsequent reactions; excessive dryness will cause the magnetic beads to crack and reduce the purification yield. Normally, drying at room temperature for 3-5 minutes is enough to allow the beads to fully dry.

7. If needed, the purified or size-selected DNA samples eluted in 0.1× TE buffer can be stored at 4°C for 1-2 weeks or at -20°C for a month.

Library Amplification

1. The library amplification step is required when using this kit. Since the transposition reaction product is not a complete double-stranded DNA library, a complete DNA library must be generated through PCR reaction.

2. When the input DNA amount is 1 ng, 8-15 amplification cycles are recommended, and the library output of 13 cycles is about 600 ng.

Library Quality Analysis

1. The constructed libraries quality is generally analyzed by measuring the concentrations and size distributions.

2. Libraries’concentrations can be measured by fluorescent-based methods such as Qubit^®and PicoGreen^® or 

3. It is NOT recommended to use absorbance-based quantification methods such as NanoDrop^®.

4. It is recommended to use qPCR method for library quantification: fluorescent-based methods such as Qubit^®and PicoGreen^® cannot differentiate the incomplete dsDNA structures (inserts with no adapter or with only one of the ends ligated with adapter) from the complete libraries. The qPCR method will only amplify and measure the complete libraries with both ends ligated with adapters (the sequencable libraries), thus providing a more accurate measurement for loading.

5. The size distribution of libraries can be analyzed using Agilent Bioanalyzer or other devices based on theprinciples of capillary electrophoresis or microfluidics.

Other Materials

1. DNA purification magnetic beads:NGS DNA Selection Beadsor AMPure XP Beads or other equivalent products.

2. Library quality analysis: Agilent Technologies 2100 Bioanalyzer or other equivalent products.

3. N5 (N5XX) and N7 (N7XX) Index Primers: NGS Tagment Index Kit for Illumina.

4. Other materials: absolute ethanol, sterile ultrapure water, TE Buffer (10 mM Tris-HCl，pH 8.0-8.5+1 mM EDTA)low retention pipette tips, PCR tube, magnetic stands, thermal cycler, etc.