Results produced from the instrument are not for use in diagnostic procedures. Apr 06, 2000 applications such as northern blot analysis, cdna library construction and cdna subtraction are demanding when it comes to starting material. The ssh assay was performed using the pcrselect cdna subtraction kit clontech, usa. This kit contains two separate protocols, allowing you to choose a method based on your starting material. Total rna isolated from human placenta was used as template rna in reverse transcription reactions using the allinonetm firststrand cdna synthesis kit together with the oligodt primer. This particular kit also includes 5 tubes of rnase inhibitor 200 l each. Download scientific diagram pcrselect cdna subtraction analysis.
Protoscript first strand cdna synthesis kit features two optimized mixes, mmulv enzyme mix and mmulv reaction mix. Plant rna extraction kit aidlab, china according to the manufacturers instructions. Energicscript first strand cdna synthesis kits for qpcr user. Following rsa i digestion, 380 ng of the ds cdna was. Clontech pcr select cdna subtraction kit user manual 0516. Use aerosolfree pipette tips for dispensing small volumes or sterile disposable pipettes for larger.
The smarter race cdna amplification kit is an improved version of our. Suppression subtractive hybridization technique ssh is a highly sensitive pcr based cdna subtraction method 16 used to identify. Plasmid dna templates for sequencing were prepared with the miniprep kit qiagen. The open qpcr instrument is intended for research use only ruo. Backgroundsubtracted element signals were used to calculate cy3cy5 ratios. The ion ampliseq library kit plus includes reagents to prepare barcoded libraries manually from dna or rna for sequencing on the ion pgm, ion proton, and ion s5ion s5 xl systems. Usb firststrand cdna synthesis kit for realtime pcr tested. The pcmvscript xr cdna library construction kit is designed for generating directional cdna libraries in. Comparative transcriptional profiling analysis of the two daughter cells from tobacco zygote reveals the transcriptome differences in the apical and basal cells. Clontech pcrselect bacterial genome subtraction kit user manual. Pcrscript amp cloning kit 3 introduction the stratagene pcrscript amp cloning kit is a polymerase chain reaction pcr cloning method that can be performed in 1 hour without adding bases to the primers. Tissuewide expression profiling using cdna subtraction.
Download scientific diagram schematic diagram of suppression subtractive. The exactstart fulllength cdna library cloning kit is functiontested in a control reaction. Kit components and storage conditions upon receiving the teloprime kit, remove the smaller inner box and store it in a 20 c freezer. Invitrogen superscript doublestranded cdna synthesis kit. Suppression subtractive hybridization method for the identification of. A subtracted cdna library from the zebrafish danio rerio embryonic inner ear.
A subtracted cdna library identifies genes upregulated during. The pcr select cdna subtraction kit clontech allows comparison of two populations of mrnas by. Smartseq v4 ultra low input rna kit for sequencing user manual 040215. Rapid amplification of cdna ends race is a technique used in molecular biology to obtain the full length sequence of an rna transcript found within a cell. Smarter race cdna amplification kit user manual iii. A modified protocol of the pcr select cdna subtraction kit was used clontech. Highcapacity cdna reverse transcription kit with rnase inhibitor. Truseq small rna library prep kit reference guide 15004197.
This kit integrates our marathon cdna amplification kit chenchik et al. The rest of the kit components should be stored at room temperature and protected from light. Messenger rnas located in myelin sheath assembly sites. Mmulv enzyme mix combines mmulv reverse transcriptase and murine rnase inhibitor, while mmulv reaction mix contains dntps and an optimized buffer.
The ssh assay was performed using the pcrselect cdna subtraction kit clontech, usa following the manufacturers instructions. High performance and complete kit for 2step realtime rtpcr, for. Sh cdnas from ctls cdnas, respectively, using the pcr select cdna subtraction kit user manual pt11171, clontech laboratories, inc. Clontech pcrselect cdna subtraction kit certificate of. A solution to this problem can be found in clontechs smart cdna synthesis kit. Protocol for exactstart fulllength cdna library cloning kit. The hiv pcr detection kit is designed to detect human immunodeficiency virus type 1 hiv1 nucleic acids in human blood plasma. Master mix 5 ml, openarray real time pcr accessories kit enough to run 10 plates 4458070 taqman openarray digital pcr, 3 pack requires additional purchasesee below includes 3 openarray digital pcr plates, 2x taqman openarray digital pcr master mix 1. Example diagram of generating a quadplex multicistronic grna cassette using the multiplex grna cloning kit. Superscript vilo cdna synthesis kit qpcr step 4 youtube. The cdna in which specific transcripts are to be found is referred to as tester and the reference cdna is referred to as driver.
The kit must produce 107 colonies from 250 ng of the exactstart rna control template when using epicentres transformax ec100 electrocompetent e. Method the implemented method is based on viral rna reverse transcription followed by pcr amplification of the obtained cdna. Improving cdna yield using superscript iii debra ann nickson. Pdf comparative transcriptional profiling analysis of. Extraction of differential expressing aphidresistance. Profiling of differentially expressed genes using suppression. Suppression subtractive hybridization technique ssh is a highly sensitive pcrbased cdna subtraction method 16 used to identify. Smartseq v4 ultra low input rna kit for sequencing user manual. The titer of ssh library and the recombination efficiency were.
Energicscript first strand cdna synthesis kits user manual description the energicscript cdna synthesis kit includes all the necessary reagents for cdna synthesis to be used in qpcr. Race results in the production of a cdna copy of the rna sequence of interest, produced through reverse transcription, followed by pcr amplification of the cdna copies see rtpcr. The high capacity cdna reverse transcription kit with rnase inhibitor contains all components necessary for the quantitative conversion of up to 2 g of total rna to singlestranded cdna in a single 20 l reaction. This means that you obtain the same relative representation. Affinityscript multiple temperature cdna synthesis kit.
The incorporation of bd smart technology also permits. Which is the best software to design cdna primers and where is it available. Free online and offline, for mac and windows, very user friendly and intuitive. The subtracted and unsubtracted cdna samples were amplified using nested pcr. This novel technology combines the performance of superscript ii reverse transcriptase with the gateway technology. Produce fulllength cdna with the transcriptor first strand cdna synthesis kit. The open qpcr user manual serves to provide a comprehensive understanding of the setup, operation and maintenance of the open qpcr system. The suppression subtractive hybridization ssh library was constructed according to the instructions of the pcrselect cdna subtraction kit.
Pcrscript amp cloning kit artificial gene synthesis. Simple, streamlined wholegenome sequencing library preparation that provides accurate and comprehensive coverage of. Introduction continued smart cdna synthesis for cdna subtraction the clontech pcr select cdna subtraction kit cat. The synthesized cdna was then used to amplify different gene regions by. In numbered lanes except lane 3, reverse transcription was performed on 2 g total rna from human skeletal muscle research samples, using the kits anchored oligo dt 18 primer.
Contents and storage requirements for the truseq rna access library prep kit. Identification of upregulated genes of bacillus amyloliquefaciens b55 during the early stage of direct surface contact with rice r109 root. Efficient cdna synthesis by allinone firststrand cdna synthesis kit. Before use, check the contents of cb1, cb2, and cw. Highsensitivitydna kit1perlibrary novextbegels,6%,10well. Order, and community panels, and incorporates barcoded. Bd smart race cdna amplification kit user manual i. In a subsequent pcr, various fragments of the dystrophin. Construction of a complementary dna cdna subtraction library. Truseq dna library preparation kit v2, set a 12 indexes, 48 samples fc1212001 truseq dna library preparation kit v2, set b 12 indexes, 48 samples fc1212002 for cluster generation on cbot and sequencing on the hiseq 2000 and hiscansq truseq pairedend cluster kit v3cboths 1 flow cell pe4001. Thermo scientific verso cdna synthesis kit provides robust transcription of rna to create a complete cdna pool. A subtracted cdna library contains cdna clones corresponding to mrnas present in one cell or tissue type and not present. The open qpcr instrument is intended for indoor use. The ssh library was constructed using takara pcr select cdna subtraction kit.
Introduction cdna synthesis using template switching technology the smartseq v4 ultra low input rna kit for sequencing cat. The clontech pcrselect cdna subtraction kit provides a powerful method for identifying differentially expressed cdnas diatchenko et al. The reactions described in the superscript doublestranded cdna synthesis kit protocol are designed to. The verso system achieves robust and sensitive reverse transcription through the combination of a high affinity rt enzyme, a unique rna priming method, and an optimized buffering system.
With this software, we performed gridding, automated spot detection, manual and automated flagging, as well as background subtraction and normalization. Clontech pcrselect cdna subtraction kit user manual. Sureselect strandspecific rna library prep for illumina. Advantages of the affinityscript multiple temperature cdna synthesis kit robust cdna synthesis across a broad range of temperatures from 3755c high affinity for primertemplate complexes promotes optimal cdna yield produces excellent yields of amplifiable cdna. The protocol in the user manual was slightly modified to adjust the. Clontech pcrselect differential screening kit user manual. Clontech pcr select cdna subtraction kit user manual pt11171 quality control data a sample kit was tested to ensure the quality and performance of this lot. Introduction the cloneminer cdna library construction kit is designed to construct highquality cdna libraries without the use of traditional restriction enzyme cloning methods. A piwi homolog is one of the proteins expressed exclusively during. Sureselect strandspecific rna library prep for illumina multiplexed sequencing mrna library preparation protocol protocol version e0, march 2017 sureselect platform manufactured with agilent sureprint technology.
Construction and analysis of the cdna subtraction library of yeast. Which is the best software to design cdna primers and where. To perform the subtraction reactions, sh and ctls cdnas were digested with rsai to obtain shorter, bluntended molecules suitable for adaptor ligation and optimal for subtractive. Differentially expressed cdnas were amplified by nested pcr, cloned into pmd18t vector, transformed into e. Construction of suppression subtractive hybridization cdna. Pdf identification of upregulated genes of bacillus. Usb firststrand cdna synthesis kit for realtime pcr tested user friendly product number 75780 this kit is specifically tailored for the synthesis of firststrand cdnas prior to realtime pcr applications. Ion ampliseq library kit plus thermo fisher scientific. Either total rna, mrna, viral rna or in vitro transcribed rna can be used as a template for reverse transcription. Pdf a subtracted cdna library from the zebrafish danio. Simple, reliable, and trouble free the superscript doublestranded cdna synthesis kit offers a simple, reliable, and trouble free method by which rna templates can be converted to cdna for use in cloning and library construction experiments. Apr 12, 2011 the superscript vilo cdna synthesis kit is designed to increase cdna yields and improve the dynamic range of your qrtpcr assays.