The StepWise DNA Library Preparation Kit is designed to generate high-quality next generation sequencing (NGS) DNA libraries compatible with Illumina(R) platforms. Starting from fragmented input-DNA (500 pg - 1000 ng), the kit contains all the enzymes and buffers for end-repairing, dA-tailing, adapter ligation and library amplification. Due to the flexible protocol, the kit can be conveniently used for construction of PCR-free libraries from 100 ng of high-quality fragmented genomic DNA, or 10 ng of circulating cell-free DNA (cfDNA). PCR primers provided in the kit are optional for users who prefer self-selected high-fidelity DNA polymerases in the library amplification.
The following workflow illustrates the processes of the Rapid DNA Lib Prep Kit.
|Tube name||8 RXN||24 RXN||96 RXN|
|End Preparation||End Prep Buffer||130 μl||338 μl||1.3 ml|
|End Prep Enzymes||30 μl||78 μl||300 μl|
|dA-Prep||dA-Prep Buffer||490 μl||1274 μl||4.9 μl|
|dA-Prep Enzymes||10 μl||26 μl||100 μl|
|Adapter Ligation||Ligase MM||255 μl||663 μl||2.55 mL|
|Ligase Mix||20 μl||52 μl||200 μl|
|Amplification||2X PCR Master Mix||250 μl||650 μl||2.5 mL|
|10X PCR Primers||40 μl||130 μl||500 μl|
|Low-EDTA TE||1 ml||3 ml||10 ml|
|PEG/NaCl Solution||1 ml||3 ml||10 ml|
The Low-EDTA TE buffer and PEG/NaCl solution can be stored at room temperature or 4°C.
All other components should be stored at -20°C.
The shelf lives of all reagents are one year when stored properly.
Required Materials Not Included
Mechanical or enzymatic methods for DNA fragmentation
Multiplex adapters (cat. no. RK20282, RK20283, RK20284, RK20285, RK20292, RK20293, RK20294, RK20295) compatible with Illumina(R) platforms
100% ethanol (ACS grade)
PCR strip tubes or plates
AgencourtTM AMPure XP bead (Beckman Coulter Inc., cat. no. A63880)
Pipettes and multichannel pipettes
Aerosol resistant pipette tips
Agilent Bioanalyzer or comparable method to assess the quality of DNA library
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