The PCR-free NGS DNA Library Prep Kit was developed for construction of DNA libraries for next generation sequencing (illumina platform). The kit adds 3′-dT-tailed library adapters to both ends of DNA fragments efficiently. The kit uses double strand DNA fragments (blunt and/or sticky) as input DNA for NGS library construction, and is compatible with DNA fragments generated from both enzymatic methods (BioDynami DNA fragmentation enzymes etc.) and physical methods (sonication, nebulization etc.).
PCR-Free NGS DNA Library Prep Kit Workflow
PCR amplification is a standard step for library preparation of Next-Generation Sequencing. The PCR is used to amplify fully ligated DNA fragments and to add index information to the libraries. The indexing is for the pooling of the library samples in an effort to reduce the sequencing cost.
However, PCR introduces uneven amplification of DNA in some DNA regions with extreme GC-contents and secondary structures. This bias can cause very low sequencing coverage in such regions. DNA sequencing in these regions is still a huge challenge.
PCR-free library prep can reduce library bias and minimize sequencing gaps. The sequencing data from PCR-free library samples have even genomic coverage with few gaps and better depth in GC-rich regions. Our PCR-free NGS kit offers optimal coverage in the regions that are traditionally difficult, such as high-GC content regions, low-GC content regions, and repetitive sequence regions.
Two index types are available for the kit:
Non-index: Libraries do not have index.
Index: Each library contains one i5 index and one i7 index. Library multiplexing up to 96 samples is possible. List of indexes can be downloaded Here.
Kit advantages:
The PCR-free NGS DNA Library Prep Kit was developed for construction of DNA libraries for next generation sequencing (illumina platform). The kit adds 3′-dT-tailed library adapters to both ends of DNA fragments efficiently. The kit uses double strand DNA fragments (blunt and/or sticky) as input DNA for NGS library construction, and is compatible with DNA fragments generated from both enzymatic methods (BioDynami DNA fragmentation enzymes etc.) and physical methods (sonication, nebulization etc.).
This kit provides a rapid method for the isolation and purification of both cytoplasmic and nuclear RNA from cultured animal cells and small tissue samples. The kit can be used to isolate all sizes of RNA from the cytoplasmic and nuclear RNA fractions, including all small RNA species without any requirement for phenol. Included in the kit are sufficient reagents to perform either 50 cytoplasmic RNA preparations or 25 cytoplasmic and 25 nuclear RNA preparations. Ten samples can be processed in approximately 45 minutes. This kit is also available in a 100 prep size.
Background
In certain circumstances it is desirable to be able to isolate fractionated RNA as opposed to total RNA. For example, it may be preferable to isolate only mature, cytoplasmic RNA for some studies on expression profiling. Alternatively it may be desirable to isolate nuclear RNA in order to investigate and study pre-processed (non-spliced) RNA. Furthermore, this kit can be used to isolate RNA for downstream applications where it is necessary to avoid DNA contamination, since the cytoplasmic fraction has been shown to be free of all traces of genomic DNA.
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| Kit Specifications | |
| Maximum Column Binding Capacity | Up to 50 µg RNA |
| Maximum Column Loading Volume | 650 µL |
| Size of RNA Purified | All sizes, including small RNA (< 200 nt) |
| Time to Complete 10 Purifications | 45 minutes |
| RNA Yield HeLa (1 x 106) – Cytoplasmic RNA HeLa (1 x 106) – Nuclear RNA | 15 µg ≤ 3.5 µg |
Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment.
| Component | Cat. 21000 (50 preps) | Cat. 37400 (100 preps) |
|---|---|---|
| Lysis Buffer J | 20 mL | 2 x 20 mL |
| Buffer SK | 40 mL | 2 x 40 mL |
| Wash Solution A | 38 mL | 2 x 38 mL |
| Elution Buffer E | 6 mL | 2 x 6 mL |
| Mini Spin Columns | 50 | 100 |
| Collection Tubes | 50 | 100 |
| Elution Tubes (1.7 mL) | 50 | 100 |
| Product Insert | 1 | 1 |
