Applicable to purePlex DNA Library Preparation Kit.
This kit offers speed, performance, and auto-normalization with unique dual indexes. Download the User Guide for more FAQs and information here.
What applications are recommended for the purePlex Library Preparation Kit?
The purePlex Library Preparation Kit is recommended for synthetic construct sequencing (amplicons, plasmids, etc.), low-pass whole genome sequencing, whole small genome sequencing (<50 Mb), single cell RNA-seq (scRNA-seq), and metagenomics/microbiome sequencing.
How many samples can be processed with a single kit?
A single purePlex kit provides enough reagents to process up to 96 samples.
Are all required adapters, indices, and amplification primers included in purePlex Library Preparation Kit?
Yes. The purePlex kit includes all the indexed adapters and amplification primers necessary to make dual-indexed Illumina-compatible libraries. It is worth noting that the i7 index is added by the i7-Tagging Reagent (i7-TR) and the i5 index is added by the i5-Tagging Reagent (i5-TR).
Are any additional reagents, consumables, or equipment needed?
Reagents: KAPA HiFi HotStart ReadyMix; 10 mM Tris-HCl, pH 8.0, ultra-pure water, ethanol, reagents for DNA and library QC (PicoGreen), and Illumina sequencing kits.
Consumables: 1.5 mL LoBind tubes; PCR plate, PCR strip tubes or individual tubes; pipette tips; plate seals or strip caps.
Equipment: Table-top vortex; plate centrifuge; minifuge; appropriate pipettors, magnet (suitable for 1.5 ml LoBind tube) for MAGwise bead-based purification steps; a thermal cycler, equipment for assessing library size by gel electrophoresis (BioAnalyzer, TapeStation, or Fragment Analyzer, etc.) and library concentration (fluorometer or qPCR instrument), and an Illumina sequencing system.
Are MAGwise purification beads the same as other purification beads?
MAGwise purification beads are similar to many other commercially available purification beads. The purePlex kit has been validated using MAGwise purification beads, thus it is recommended to use the included MAGwise purification beads for optimal performance. However, other SPRI purification beads should perform similarly and can be utilized/implemented with optimization.
The MAGwise purification beads were accidentally stored at the wrong temperature. Can it still be used?
To better understand the potential impact and for guidance, contact [email protected]
. Short term storage of MAGwise at room temperature (<2 weeks) or -20°C (<2 days) does not appreciably alter performance for most applications.
Coding buffer and X-solution were accidentally stored at the wrong temperature. Can they still be used?
Yes. Equilibrate the coding buffer and X-solution to room temperature by removing them from the refrigerator and/or defrosting them. Before use, ensure they are homogenous. Coding Buffer should be vortexed then pulse-fuged. X-solution should be checked for precipitate. If precipitate is present in the X-solution, warm to 37°C, then invert or pipet to mix. Do NOT vortex X-Solution. Store remaining Coding Buffer and X-Solution at room temperature.
Is there flexibility in pooling for the purePlex Library Preparation Kit?
Yes. The purePlex Library Preparation Kit workflow has been optimized and validated for 24 samples per pool, with columns 1-3 in pool A, columns 4-6 in pool B, columns 7-9 in pool C and columns 10-12 in pool D. However, because both the i7 and i5 tagging steps are performed on individual samples, the purePlex kit allows flexibility in pooling and batch sizes. Please see the User Guide for alternate pooling strategies and recommendations for best practices.
The concentration of DNA sample input is variable. Can the samples still be prepped together?
The purePlex Library Preparation Kit performs optimally with 25 ng of dsDNA per well, however, individually adjusting each sample to 5 ng/µl is not necessary as purePlex Library Preparation Kits are formulated to tolerate up to a 10-fold difference in sample input (5 to 50 ng).
Can a different index combination of i7-Tagging Reagent and i5-Tagging Reagent for the purePlex Library Preparation Kit be used?
No. The purePlex Library Preparation Kit has been optimized for the specific combination of i7-TR and i5-TR. Alternative combinations may affect autonormalization, fragment length, library concentration and complexity. However, incorrect combinations of i7-i5 tagging reagent are likely to generate sequence-able library and depending on the sequencing application, it is possible to move forward with the library preparation and sequencing without impacting the sequencing results.