Description
ER/A Tailing Enzyme Mix is an NGS library preparation module. The module uses a one-step reaction combining end-repair and dA-tailing to convert fragmented DNA into 5ʹ-phosphorylated and 3ʹ-dA-tailed DNA fragments, enabling direct ligation of Illumina sequencing adapters. When used in combination with the WGS ligation module (cat. no. L6030-W-L), the optimized chemistry ensures high sensitivity for low input DNA, high ligation efficiency for maximum library yield and a workflow that is under 3 hours with less than 45 minutes hands on time. ER/A Tailing Enzyme Mix is supplied with 10x ERA Buffer (cat. no B9420).
Enzyme properties Storage temperature: –25°C to –15°C
The optimized chemistry and protocol provide a streamlined workflow ensuring high sensitivity and efficiency for a wide range of DNA iputs, while maximizing library yields. The ER/A-Tailing Enzyme Mix offers a flexible library solution to address quality, speed, and throughput while remaining a cost -effective option for small and large sequencing operations. Compatible with gDNA, cDNA, and FFPE/FF samples
Enter the following program into a thermal cycler (see table below). Be certain to use the instrument’s heated lid, and if possible, set the temperature of the heated lid to 70°C. When the thermal cycler block reaches 4°C, pause the program. Compatible with DNA inputs as low as 250 pg and up to 1µg in water, EB or 1X TE Step Incubation temperature (°C) Incubation time (minutes) 1 4 1 2 20 30 3 65 30 4 4 Hold It is important to follow the procedure described below to achieve optimal results. Prepare a reaction mix in a new, thin-walled PCR tube on ice by combining ERA Buffer, DNA sample and nuclease-free water as indicated in the table for each DNA sample. Mix well by gently pipetting (do not vortex to mix). The final reaction volume is 50 µl. Volume for one reaction (µl) 10X ERA Buffer 5 DNA sample X Nuclease-free water (35–X) Total 40 3. Add 10 µl of 5X ER/A-Tailing Enzyme Mix to each reaction and gently mix well by pipetting up and down 6–8 times. It is recommended to keep the PCR tube on ice during the entire reaction setup. 4. Pulse-spin the sample tube and immediately transfer to the pre-chilled thermal cycler (4°C). Resume the cycling program. 5. When the thermal cycler program is complete and the sample block has returned to 4°C, remove samples from block and place on ice. 6. Proceed directly into Adapter Ligation. We recommend using WGS Ligase (cat. no. L6030-W-L) Quality control analysis ER/A Tailing Enzyme Mix Functional Assay: Quality control (QC) library length must be within 15% of the reference library length. Concentration of the QC library generated from 100 ng input DNA (average approximately 300 bp fragments) is >60 nm with mapped reads >90%. For QC library, normalized coverage should be within 0.7 to 1.3 for most of the genome (10–80% GC content). Enzyme components were tested prior to assembly and free of contaminating endonucleases and exonucleases. Enzyme purity was >95% as determined by SDS-PAGE and negligible E. coli genomic DNA contamination was confirmed by qPCR.
