Sharpvue™ Gene First Strand Kit
Contents
1. 50 C for 3 hours Use DEPC to treat all microcentrifuge tubes pipettes tips if not RNase free and then autoclave to deactivate RNase RNase free consumable are available for purchase from many commercial sources IMPORTANT NOTES 1 Store kit at 20 C Avoid storage or leaving reagents at 4 C or room temperature 2 Touch gently to avoid shocking severely then briefly centrifugr before use 3 Setup all reactions on ice to reduce risk of RNA degradation 4 Read all procedures before setting up RT reaction Procedure 1 Reverse transcription of mRNAs into single stranded cDNAs by using Sharpvue Gene First Strand Kit a Set the following components on ice in a tube Component Volume ul RNA sample X Sharpvue Gene First Strand Kit 5x Mix A Cat No 9000002 2 Sharpvue Gene First Strand Kit 40x Mix B Cat No 9000003 0 25 Sharpvue Nuclease Free Water Cat No 9000016 to 10 For Multiple reactions prepare a reaction mix of common components b Mix gently and spin the tube briefly to collect the contents c Transfer the tube to a thermal cycler Incubate at 42 C for 5 60 minutes then incubate at 85 Cfor 5 minutes to inactivate the reaction d Store the single stranded cDNA at 20 C or proceed directly to PCR amplification 2 Quantifying cDNAs with real time PCR by using forward primer reverse primer and 2X Sharpvue qPCR Master Mix a Set the following components in a tube o2. ate or secondary structure of the amplification product is obstructing the reaction Prepare the RNA Primer Mix before the RT step Then add a PCR enhancing reagent such as DMSO betaine etc in the PCR reaction PCR product is longer than expected Genomic DNA was present Perform a DNase I digest before the RT step or design intron spanning or flanking primers to avoid co amplification of genomic DNA The wrong product was amplified Optimize the PCR reaction conditions Limited Use License and Warranty Limited Use License Following terms and conditions apply to use of all miRNAs and Packaging Kit the Product If the terms and conditions are not acceptable the Product in its entirety must be returned to Biovue within 5 calendar days A limited End User license is granted to the purchaser of the Product The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product must not be resold repackaged or modified for resale or used to manufacture commercial products without prior written consent from Biovue This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research Use of any part of the Product constitutes acceptance of the above terms Limited Warranty Biovue warrants that the Product meets the specification described in the accompanyi
3. cription High sensitivity and specificity miRNA RT Kit easy to operate Sharpvue 2x Universal qPCR Master Mix High Non toxic EvaGreen based Rox real time quantitative PCR Mix Human miRNA Assay Primer Sets covering 1700 human miRNAs from latest Human miRNA Primer Array Set v1 0 384 well miRBase release of v17 0 two forms of Human miRNA Primer Array Set v1 0 96 well design are 5 and 20 plates Sharpvue Gene Expression Assay Accurate quantification of mRNA expression Sharpvue 2x Universal qPCR Master Mix High Non toxic EvaGreen based Rox real time quantitative PCR Mix Gene First Strand Kit Contents and Storage Contents Quantity laces 5x MixA 25 reacction 20 C 40xMixB 25 reacction 20 C dd H2O RNase and DNase free iml Room temperature Preparation Wearing a lab coat disposable gloves and protective goggles are recommended when handling chemicals RNA Sample Preparation When working with RNA it is important to avoid RNases in your solutions consumables and labware When preparing your RNA samples always wear a mask and disposable gloves in all procedures Follow the described procedures you are using for RNA extraction carefully Ready to use solutions that are RNase free can be purchased Alternatively treat solutions with diethyl pyrocarbonate DEPC and then autoclave RNases on labware can also be inactivated by DEPC treatment or by baking at 2
4. ng Product Datasheet If it is proven to the satisfaction of Biovue that the Product fails to meet these specifications Biovue will replace the Product In the event a replacement cannot be provided Biovue will provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to Biovue 30 days of receipt of the Product Biovue s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price Biovue s liabilitydoes not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty Biovue does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose Biovue is committed to providing our customers with high quality products If you should have any questions or concerns about any Biovue products please contact us at 86 21 34612632 Biovue Technology Shanghai Ltd 2nd Floor 1st Building 2140 XieTu Road Shanghai 200032 China www biovuetech com Tel 86 21 34612632 Fax 86 21 64183997 E mail support Biovuetech com
5. r in a PCR plate Components Volume ul Forward Reverse primer mixture each 3 33x 3 RT product from d 0 67 Sharpvue 2x Universal qPCR Master Mix Cat No 9000007 5 Add Nuclease free H2O to 10 Reaction volume could be proportionally changed to 5 to 50 ul b Cap the tube or seal the plate with optical film Mix gently then spin the tube briefly to collect the contents c Set the thermal profile as follows Cycling Step Temperature HoldingTime peg Enzyme hot activation 96 C 2 minutes 1 Denaturation 96 C 5 seconds 40 Annealing 60 C 20 60 seconds 96 C 15 seconds Melting analysis 60 C 1 minutes 1 96 C 15 seconds d Transfer the tube or the plate to a real time thermal cycler 3 Real time measurement of the fluorescence change of EvaGreen fluorescent dye Trouble Shooting Guide Little for no RT PCR product RNA template degradation The quality of RNA is the key factor for cDNA synthesis Follow the RNA isolation kit procedure carefully always wearing a lab coat gloves and maskwhen working with RNA and use RNA Grade reagents and materials Check the RNA quality by RNA electrophoresis in a denaturing gel An inhibitor was present in the RNA template Trace amounts of inhibitor such as guanidine salts in the RNA template can inhibit the cDNA synthesis Re precipitate the RNA with ethanol and wash the pellet with 75 ethanol A G C rich templ
6. ss Emn 2 _ Biovue Technology Sharpvue Gene First Strand Kit For reliable first strand cDNA synthesis from all mRNA sources Cat No 9000001 25 reactions User Manual I Sharpvue miRNA First Strand Kit Sharpvue Gene Expression Assay an EvaGreen based real time quantitative PCR method for gene expression detection Description Biovue high performing gene expression assay is based on Biovue s proprietary microTaq DNA polymerase and EvaGreen dye MicroTaq DNA polymerase is inactive in its original form and become highly active and highly specific after two minute incubation at 95 C This assay has several advantages First primer dimer formation is greatly diminished Second reactions could be accomplished very quickly each cycle may just take less than 30 seconds Assay could be done with 2 tube protocol Unique among the similar products on the market our assay system employs EvaGreen After cycling the amplicon could be subjected to high definition melting analysis giving researchers more definitive answer to the purity of their product Also the high definition melt could be used to detection SNP or mutation in expressed MRNA Biovue respects researchers human kind and our environment All components in our Sharpvue Gene Expression Assay system are friendly to human and environment Neither mutagens nor cytotoxic agents are used Related Products Sharpvue miRNA Assay Product Name Des
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