No, libraries made with SeqPlex™-I WTA Kit fit directly into NGS workflows. Sequencing instrument operators should be notified of running libraries created with SeqPlex™-I WTA Kit and to expect a slight signal from any remaining primers in the IVC plots.
SEQRI
SEQPLEX-I WTA Kit
Whole Transcriptome Amplification, RNA Amplification
別名:
RNA amplification kit
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| あなたへ/カタログ番号 | 在庫状況 | 単価 |
|---|---|---|
24 reactions | カートの在庫状況を確認する | ￥202,000 |
96 reactions | カートの在庫状況を確認する | ￥681,000 |
384 reactions | カートの在庫状況を確認する | ￥2,410,000 |
この商品について
NACRES:
NA.55
UNSPSC Code:
41121800
￥202,000
カートの在庫状況を確認する
technique(s)
whole genome amplification: suitable, whole transcriptome amplification: suitable
dilution
(WTA)
input
purified RNA
compatibility
Illumina (Next Generationa Sequencing)
shipped in
wet ice
storage temp.
−20°C
General description
The SeqPlex™-I whole transcriptome amplification (WTA) kit allows the amplification of small quantities of reverse transcribed RNA or degraded RNA for direct input onto Illumina® next-generation sequencing (NGS) flow cells. The SeqPlex-i process is comprised of three steps:
Pre-amplification/Library Synthesis: In the Pre-amplification/Library Synthesis step using the (Library Preparation Reagents), the template RNA is reverse transcribed using primers composed of a semi-degenerate 3′- and universal 5′-ends. As polymerization proceeds, displaced and RNaseH generated single strands serve as new templates for additional primer annealing and extension producing random, overlapping cDNAs flanked by a universal primer (5′) and primer complement (3′) sequence.
Amplification 1: In the Amplified Library Synthesis step (using the Amplification 1 Reagents), products from pre-amplification/library synthesis are amplified by single primer PCR via the proprietary universal end sequence. These amplification products typically range from 200 to 500+ base pairs. Note: Amplicons from degraded RNA, such as Formalin Fixed Paraffin Embedded (FFPE), are typically shorter and dependent upon the length of the starting RNA.
Amplification 2: In the Sequencing Library Synthesis step (using Amplification 2 Reagents), single primer amplicons from amplification 1 are converted to dual Illumina® primer PCR products ready for purification, quantification, and Illumina® NGS.
Pre-amplification/Library Synthesis: In the Pre-amplification/Library Synthesis step using the (Library Preparation Reagents), the template RNA is reverse transcribed using primers composed of a semi-degenerate 3′- and universal 5′-ends. As polymerization proceeds, displaced and RNaseH generated single strands serve as new templates for additional primer annealing and extension producing random, overlapping cDNAs flanked by a universal primer (5′) and primer complement (3′) sequence.
Amplification 1: In the Amplified Library Synthesis step (using the Amplification 1 Reagents), products from pre-amplification/library synthesis are amplified by single primer PCR via the proprietary universal end sequence. These amplification products typically range from 200 to 500+ base pairs. Note: Amplicons from degraded RNA, such as Formalin Fixed Paraffin Embedded (FFPE), are typically shorter and dependent upon the length of the starting RNA.
Amplification 2: In the Sequencing Library Synthesis step (using Amplification 2 Reagents), single primer amplicons from amplification 1 are converted to dual Illumina® primer PCR products ready for purification, quantification, and Illumina® NGS.
To order SeqPlexI adapters in tubes, please download NGSO Adapters SeqPlexI tubes (XLSX File). To order arrayed adapters in plates, download NGSO Plate SeqPlexI arrayed plates (XLSX File). Details on our custom adapters product, Next-Gen Sequencing Oligos (NGSO), can be found at SigmaAldrich.com/nextgenoligos. From this page, you can directly access an online ordering configurator by clicking on the "Order Now" option. Both spreadsheets are directly uploadable after selecting the quantity and/or format from the dropdown menus. For adapters in solution in tubes or in plates, NGSO-Silver is the only option available, which has a length cutoff of 60 bases (several of the adapters are longer than 60 bases). In addition, adapters in plates cannot be duplexed. If you would like a feasibility assessment of ordering NGSO-Silver longer 60 bases or duplexed in plates, then please send a request to [email protected]. All of the adapters are readily available for online ordering if dry and in tubes as either NGSO-Bronze or Gold (recommended purification for any adapter).
Application
SeqPlex™-I WTA Kit has been used for whole transcriptome amplification. It has also been used to sequence the total nucleic acid from chikungunya virus (CHIKV) cytopathic effect (CPE) positive cell culture samples.[1]
Features and Benefits
- Amplifies fragmented/extremely small quantities of total RNA: Fragmented or intact RNA from all sources including FFPE and RIP are easily amplified by random priming technology.
- Semi-degenerate library primer design ensures more complete transcriptome coverage and efficient priming
- Fewer Steps: No need to fragment cDNA before sequencing
- High-efficiency: Amplifies ds-cDNA in 8 hours or less
- Cost-effective: No longer requires an additional NGS library prep step
- Compatible with Illumina® next generation sequencing
Other Notes
1) RNA Handling Technique
a) The reagents in this kit have been tested to assure that RNases are absent.
b) The user, however, must protect the integrity of experimental results by wearing basic protective equipment, including gloved hands and lab coat.
c) All reagent transfers throughout this procedure should be performed in a laminar flow hood or dedicated clean room.
d) Frozen RNA samples should be thawed on ice.
2) A 20 μL Amplification 2 reaction will produce >100 ng of amplified double-stranded cDNA when starting with 100 pg to 5 ng of high-quality RNA. Higher input quantities and higher quality RNA template generally result in increased yields. For damaged RNA, such as from FFPE, 1–50 ng input RNA is recommended.
3) The dual index adapter primers (AP100) provided in this kit will only work for one sample. If pooling samples for sequencing is required, the user must provide additional index primer sets. See example index primer sequences on page 2 of the technical bulletin.
a) The reagents in this kit have been tested to assure that RNases are absent.
b) The user, however, must protect the integrity of experimental results by wearing basic protective equipment, including gloved hands and lab coat.
c) All reagent transfers throughout this procedure should be performed in a laminar flow hood or dedicated clean room.
d) Frozen RNA samples should be thawed on ice.
2) A 20 μL Amplification 2 reaction will produce >100 ng of amplified double-stranded cDNA when starting with 100 pg to 5 ng of high-quality RNA. Higher input quantities and higher quality RNA template generally result in increased yields. For damaged RNA, such as from FFPE, 1–50 ng input RNA is recommended.
3) The dual index adapter primers (AP100) provided in this kit will only work for one sample. If pooling samples for sequencing is required, the user must provide additional index primer sets. See example index primer sequences on page 2 of the technical bulletin.
Legal Information
Illumina is a registered trademark of Illumina, Inc.
SeqPlex is a trademark of Sigma-Aldrich Co. LLC
Disclaimer
The SeqPlex-I DNA Amplification Kit for whole genome amplification (WGA) is for R&D use only. Not for drug, household, or other uses.
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当該品目 | |||
|---|---|---|---|
| technique(s) whole genome amplification: suitable, whole transcriptome amplification: suitable | technique(s) DNA amplification: suitable, PCR: suitable, whole genome amplification: suitable | technique(s) whole genome amplification: suitable | technique(s) whole genome amplification: suitable |
| dilution (WTA) | dilution (WGA) | dilution (WTA) | dilution (WGA) |
| compatibility Illumina (Next Generationa Sequencing) | compatibility Illumina Next Generation Sequencing | compatibility - | compatibility - |
| shipped in wet ice | shipped in wet ice | shipped in wet ice | shipped in wet ice |
| input purified RNA | input purified DNA | input purified RNA | input purified DNA |
| storage temp. −20°C | storage temp. −20°C | storage temp. −20°C | storage temp. −20°C |
signalword
Danger
hcodes
pcodes
Hazard Classifications
Resp. Sens. 1
保管分類
10 - Combustible liquids
wgk
WGK 3
適用法令
試験研究用途を考慮した関連法令を主に挙げております。化学物質以外については、一部の情報のみ提供しています。 製品を安全かつ合法的に使用することは、使用者の義務です。最新情報により修正される場合があります。WEBの反映には時間を要することがあるため、適宜SDSをご参照ください。
Please refer to KIT Component information
pdsc
Please refer to KIT Component information
prtr
Please refer to KIT Component information
fsl
Please refer to KIT Component information
ishl_indicated
Please refer to KIT Component information
ishl_notified
Please refer to KIT Component information
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キットコンポーネントの情報を参照してください
jan
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Will SeqPlex™-I libraries require special NGS sequencing protocols?
1 回答-
役に立ちましたか？
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Will reducing cycles during amplification improve representation?
1 回答-
No, you need to reach “plateau” for optimum representation. Proceeding past 1-2 cycles will not negate the reactions, but best representation is achieved around “plateau”. Insufficient cycling leads to a significant reduction in representation/coverage.
役に立ちましたか？
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Are there advantages to SeqPlex™-I over GenomePlex?
1 回答-
SeqPlex™-I Pre-Amplification primers have been designed to target more frequently than existing GenomePlex WTA primers and therefore may provide the advantage of superior genome coverage in some regions.
役に立ちましたか？
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Is SeqPlex™-I WTA Kit compatible with microarrays and qPCR?
1 回答-
Yes, libraries made using SeqPlex™-I WTA Kit can be used in these applications like genomic DNA or existing GenomePlex products.
役に立ちましたか？
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