Description
WGS Fragmentation Mix provides a single-tube solution for library construction for Illumina platforms. The protocol supports fragmentation, end-repair and dA-tailing in a single reaction step, greatly simplifying the workflow and reducing the total reaction time and hands-on time. WGS Fragmentation Mix is supplied with 10X Fragmentation Buffer (cat. no. B0330) and Enhancer (cat. no. B0340).
Genome coverage of WGS Fragmentation Mix is comparable to mechanical shearing with both 100 ng and 1 ng of DNA WGS Fragmentation Mix provides: Faster workflow with high-quality, reproducible libraries Higher library yield and better sensitivity at 100 ng and 100 pg input Customizable fragment sizes and highly reproducible fragmentation Lower base distribution bias at starting position Significantly lower duplication rate for low DNA inputs Performance from any input Enzyme properties: Storage temperature: –25°C to –15°C
General Precautions Use good laboratory practices to minimize cross-contamination of nucleic acid products. Always use PCR tubes, microfuge tubes and pipette tips that are certified sterile, DNase- and RNase-free. Before starting, wipe down work area and pipettes with an RNase and DNA cleaning product, such as RNase Away (Molecular BioProducts, Inc. San Diego, CA). For consistent library amplification, ensure the thermal cycler used in this protocol is in good working order and has been calibrated according to the manufacturer’s specifications. Read the entire protocol before beginning. Take note of stopping points where samples can be frozen at –20°C and plan your workflow accordingly. Enzyme-based DNA fragmentation is sensitive to many factors, such as reaction temperature, time, and setup conditions, as well as the input DNA. We strongly recommend users practicing the protocol and optimizing the parameter (reaction time) using the same or similar DNA samples. Before you begin Thaw reagents on ice. Once reagents are thawed, mix the 5X Fragmentation Enzyme Mix by finger flicking (do not vortex to mix), and mix other components by quick vortexing to avoid any localized concentrations.
Before setting up the reaction, it is critical to determine the amount of the input DNA as well as the buffer that the DNA is in. We strongly recommend users practice this protocol and optimize the reaction time using the same or similar DNA samples. Prepare the following program into a thermal cycler (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. The input DNA (1–1000 ng) is free of EDTA or in EB or 0.1X TE Step Incubation temperature (°C) Incubation time (minutes) 1 4 1 2 32 3–24 3 65 30 4 4 Hold Guidelines for choosing the initial fragmentation time (minutes) at 32°C Input DNA, ng Fragment peak size 250 bp 350 bp 450 bp 550 bp 10 24 16 14 10 100 16 10 8 6 1000 14 8 6 4 * Note: The exact reaction time may need to be optimized for different amounts of input DNA. The table above serves as a general guideline to help decide the starting point for optimization of the reaction time to achieve the desired fragment size. For initial optimization, we recommend including two additional time points, one 3 minutes longer and another one 3 minutes shorter. Fine-tuning may be required if precise fragment size is critical. For input DNA ≤10 ng: to produce fragment size centers around 350 bp, we recommend adding 2.5 µL Enhancer to a 50 µL reaction and incubate for 10 minutes. 3. It is important to follow the procedure described below, to achieve optimal results. The final reaction volume is 50 µl: use the table below to set up the reaction. Prepare a master mix on ice by combining Fragmentation Buffer, DNA, and nuclease-free water as indicated in the table (volumes are for one DNA sample). Mix well by gently pipetting (do not vortex to mix). The mix can be scaled as needed for the desired number of samples (10% extra volume added to compensate for the pipetting loss when preparing the master mix for multiple samples). Volume per one reaction (µl) For >10 ng input DNA For 1–10 ng input DNA 10X Fragmentation Buffer 5 5 Purified DNA X X Enhancer 0 2.5 Nuclease-free water (35–X) (32.5–X) Total volume 40 40 4. Transfer 10 µl 5X WGS Fragmentation Mix to a new, thin-walled PCR tube for each reaction. Add 40 µl master mix from step 3 and gently mix well by pipetting up and down 10–12 times. It is critical to keep the PCR tube on ice during the entire reaction setup. 5. Pulse-spin the sample tube and immediately transfer to the pre-chilled thermal cycler (4°C). Resume the cycling program. 6. When thermal cycler program is complete and the sample block has returned to 4°C, remove samples from block and place on ice. 7. Immediately proceed to the ligation step. To achieve optimal ligation efficiencies, we recommend using WGS Ligase (cat. no. L6030-W-L) and if needed use HiFi PCR Master Mix (cat. no. P7670). Quality control analysis The functionality of the 5X WGS Fragmentation Mix is evaluated by performing library construction, PCR/qPCR and sequencing. Enzyme components were tested prior to assembly and found 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. Specific lots of Enzyme Mix (cat. no. Y9410) and 10X Fragmentation Buffer (cat. no. B0330) are tested and released together. Performance is not guaranteed if enzyme and buffer are purchased separately.
