Cat. No. / ID: Y9410L
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 as well as the hands-on time.
WGS Fragmentation Mix is supplied with 10X Fragmentation Buffer (cat. no. B0330) and Enhancer (cat. no. B0340).
SDS available upon request
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.
|Step||Incubation temperature||Incubation time|
|Input DNA, ng||Fragment peak size|
|250 bp||350 bp||450 bp||550 bp|
* 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).
|For >10 ng input DNA||For 1–10 ng input DNA|
|10X Fragmentation Buffer||5||5|
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.
This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.