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.
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추천 제품
기술
whole genome amplification: suitable
whole transcriptome amplification: suitable
호환성
Illumina (Next Generationa Sequencing)
배송 상태
wet ice
저장 온도
−20°C
일반 설명
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).
애플리케이션
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]
특징 및 장점
- 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
기타 정보
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.
법적 정보
Illumina is a registered trademark of Illumina, Inc.
SeqPlex is a trademark of Sigma-Aldrich Co. LLC
면책조항
The SeqPlex-I DNA Amplification Kit for whole genome amplification (WGA) is for R&D use only. Not for drug, household, or other uses.
신호어
Danger
유해 및 위험 성명서
예방조치 성명서
Hazard Classifications
Resp. Sens. 1
Storage Class Code
10 - Combustible liquids
John T Kayiwa et al.
Tropical diseases, travel medicine and vaccines, 5, 21-21 (2019-12-05)
Arboviruses are (re-) emerging viruses that cause significant morbidity globally. Clinical manifestations usually consist of a non-specific febrile illness that may be accompanied by rash, arthralgia and arthritis and/or with neurological or hemorrhagic syndromes. The broad range of differential diagnoses
문서
SeqPlex™-I WTA kit amplifies RNA for NGS, enabling genomic studies from limited samples.
관련 콘텐츠
Major technological advances have made the production of monoclonal antibodies quicker and more efficient. There are three established platforms for antibody discovery. We offer reagents for production of monoclonal antibody libraries using each of these techniques.
최근 기술 분야에서 중대한 발전들이 이뤄지면서 단클론 항체 생성의 속도와 효율성이 더욱 높아졌습니다. 항체 발견을 위해 확립된 플랫폼은 3가지가 있습니다. Merck는 이 기법들을 각각 이용해서 단클론 항체 라이브러리를 생성하는 데 필요한 시약을 제공합니다.
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Will reducing cycles during amplification improve representation?
1 답변-
도움이 되었습니까?
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Is SeqPlex™-I WTA Kit compatible with microarrays and qPCR?
1 답변-
Yes, libraries made using SeqPlex™-I WTA Kit can be used in these applications like genomic DNA or existing GenomePlex products.
도움이 되었습니까?
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Are there advantages to SeqPlex™-I over GenomePlex?
1 답변-
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.
도움이 되었습니까?
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Will SeqPlex™-I libraries require special NGS sequencing protocols?
1 답변-
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.
도움이 되었습니까?
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