The CD Universal Adapter Ligation Module for Illumina has been configured to ligate Illumina-compatible DNA adaptors to 5-phosphorylated and 3-dAtailed DNA fragments efficiently. The kit is designed to work with Illumina's CD End Repair/dA-Tailing Module (MOD001) and is part of a universal DNA library prep workflow that can handle 100pg-1g of input DNA. The kit lowers the cost of RNA sequencing for high-throughput experiments, allowing gene expression profiling studies to be done with NGS rather than arrays. It also offers a low-cost DNA sequencing solution for whole-genome resequencing, targeted resequencing, de novo sequencing, metagenomics, and methylation studies on a large scale.
Application:
Suitable for 100pg-1μg end preparation products (5´-phosphorylated and 3´-dAtailed DNA fragments). Used for one-step DNA adaptors ligation application to DNA library preparation for Illumina platform.
Storage:
The Kit should be stored at -30~-15°C.
Components:
Specifications:
| Sample amount | 100pg-1μg |
| Application | Suitable for 100pg-1μg end preparation products (5´-phosphorylated and 3´-dAtailed DNA fragments). Used for one-step DNA adaptors ligation application to DNA library preparation for Illumina platform. |
| Sample type | End preparation products (5´-phosphorylated and 3´-dAtailed DNA fragments) |
| Sequencing Platform | Illumina |
This process is to ligate End Preparation products to Adapters.
- Dilute adapter stocks to the appropriate concentration, as below:
- Thaw the Rapid Ligation Buffer 2 and mix thoroughly. Place on ice.
- Prepare the reaction solution in a sterile PCR tube as follows:
- Mix thoroughly by gently pipetting up and down. DO NOT Vortex! Spin down briefly.
- Put the tube in a PCR instrument and run the following PCR program (Hot Lid Temperature: 105°C)
- Libaray purification. It is recommended to use CD DNA Clean Beads (CB001) to cleanup the Adapter Ligation products. Following the CD Universal DNA Library Prep Kit for Illumina (DP001) steps.
- Library Amplification. Following the CD HiFi Amplification Mix (MOD003) steps.
Recommended adapter concentrations for libraries prepared from 100 pg - 4 µg input DNA
| Input DNA | Adapter: Input DNA Molar Ratio | Adapter concentration from other source | CD Adapter Dilution Ratio |
| 500 ng-4 μg | 10:1-20:1 | 10 μM | Undiluted |
| 100 ng-500 ng | 20:1-100:1 | 10 μM | Undiluted |
| 25 ng-100 ng | 50:1-200:1 | 5 μM | 1:2 |
| 5 ng-25 ng | 40:1-200:1 | 1 μM | 1:10 |
| 100 pg-5 ng | 60:1-3000:1 | 0.2 μM | 1:30-1:100 |
• The quality and amount of Adapters directly affect the preparation efficiency and library quality. The recommended ratio of adapter: input is between 10 : 1 and 200 : 1. High Adapter input may lead to residual Adapter/Adapter Dimer. Low Adapter input may affect ligation efficiency and reduces library yields.
• Calculate the moles of Input DNA:
moles of Input DNA (pmol) ≈ mass of Input DNA (ng) / [0.66 × average length of Input DNA (bp)]
• According to the concentration or dilution ratio, dilute Adapter with 0.1× TE. Make the volume of Adapter fixed (5 µl) to avoid pipetting error.
• The quality of adapters will affect the molar ratio of Adapter and Input DNA and further affect ligation rate and library yields. Please see the adapter with high quality for library preparation. Dilute and store the Adapter solution with 0.1× TE. Minimize the number of freeze-thaw cycles.
• Increasing adapter inputs can increase library yields, especially when the amount of Input DNA is ≤ 25 ng. When optimizing workflows, two or three adapter concentrations should be evaluated: try the recommended adapter concentration, as well as one or two additional concentrations in a range that is 2-10 times higher than the recommended concentration. If the adapter concentration is limited, try using more volume to increase adapter amount. For example, if Input DNA is 500 ng-4 µg while default volume of adapter is 5 µl, please increase to 10 µl to enhance 5% - 15% library output.
| Reagents | Volume of Sample |
| End Preparation Products | 65 µl |
| Rapid Ligation buffer 2 | 25 µl |
| Rapid DNA ligase | 5 µl |
| DNA Adapter X | 5 µl |
| Total Volume | 100 µl |
| Temperature | Time |
| 20°C | 15 min |
| 4°C | Hold |
