Hieff NGS™ OnePot Flash DNA Library Prep Kit (Enzymatic) -12316ES

Hieff NGS™ One Pot Flash DNA Library PrepKit is a rapid enzymatic DNA library kit，witch contains high quality enzyme for DNA fragmentation and combines DNA fragmentation, end-repair, and dA-tailing into one step, which reduce time significantly and cost of library preparation.

This library prep kit Compatible with 100 pg-500 ng samples of all common animals, plants, microorganisms, etc.

and quickly realize DNA fragmentation, terminal repair and A-tail addition reaction in a single tube.The kit needs to be matched with adapters and primers, and is compatible with Illumina and MGI high-throughput sequencing platforms.

Feature

1) Suitable for genomic DNA samples of 100 pg-500 ng.

2）compatible with Illumina and MGI high-throughput sequencing platforms.

3）Fragmentation, end-repair and A-tailing reaction within 5 min.

4）Efficient library conversion rate and amplification efficiency.

Specifications

Components

Note: * indicates that this reagent is not included in this kit and additional reagents are required.The kit is compatible with dual platforms of Illumina & MGI, but additional primer mix (CAT # 13334 Primer Mix for MGI and Cat# 13335 Primer Mix for Illumina) is required.

Storage

This product should be stored at -25~-15℃ for 1 year.

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 and place them on ice for later use.

3.When preparing the reaction solution in each step, it is recommended to use a pipette to blow and mix evenly or shake gently. Violent shaking may cause the library output to decrease.

4. In order to avoid cross contamination of samples, it is recommended to use a gun head with a filter element. Please replace the gun head when absorbing different samples.

5. It is recommended to perform each reaction step in a thermocycler with a heated lid. The thermocycler should be preheated to the set temperature before use.

6. Improper operations may very likely cause aerosol contaminations, impacting the accuracy of result. Mandatory physical isolation of PCR reaction mixing regions and PCR product purification assay regions is recommended. Equipped with equipment such as specialized pipettes for library construction.

7. This product is for research use only.

About DNA fragmentation

1. The compatible range of this kit is 100 pg–500 ng input DNA. High quality Input DNA with A260/A280 = 1.8-2.0 should be used as much as possible.

2. If the Input DNA contains high concentration of metal ion chelating agent or other salts, it may affect the subsequent experiments. It is recommended to dilute the DNA in ddH2O or Tebuffer (10 mm tris-HCl, pH 8.0-8.5; 0.1 mM EDTA).

3. For most high-quality genomic DNA, the digestion time is shown in Table 1. The kit has low preference and can tolerate various templates with GC content.

Table 1.  Recommended time of conventional genomic DNA fragmentation

Adapter Ligation

1. The concentration of the adapter directly affects the ligation efficiency and library yield. Excessive use of Adapter may produce more adapterdimer; Low dosage may affect the ligation efficiency and library yield. Tables 2 and 3 list the recommended amount of Adapter for different Input DNA inputs using this kit.

Table 2.  The recommended Illumina adapter amount for different input DNA

Table 3.  The recommended MGI adapter amount for different input DNA

Library Amplification

Amplification cycle numbers should be strictly controlled. Insufficient amplification may lead to low library yield; Over-amplification may introduce increased bias, errors, duplicated read, and chimeric products. Table 4 lists recommended cycle numbers targeting the library yield of 1 μg.

Table 4.  The recommended cycles of 100 pg-500 ng Input DNA

Bead-based DNA Cleanup and Size Selection

1. There are multiple steps in the library construction process that require DNA purification magnetic beads. We recommend Hieff NGS™ DNA Selection Beads (Yeasen Cat#12601) or AMPure™ XP magnetic beads (Beckman Cat#A63880) for DNA purification and size-selection.

2. 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.

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

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

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

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 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 qPCR.

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 the principles of capillary electrophoresis or microfluidics.

Manual