First-Strand cDNA Synthesis Kit For reliable first
Contents
1. Expressway to Discovery All in One First Strand cDNA Synthesis Kit For reliable first strand cDNA synthesis from all RNA sources Cat No AORT 0020 20 synthesis reactions Cat No AORT 0050 50 synthesis reactions User Manual GeneCopoeia Inc 9620 Medical Center Drive 101 Rockville MD 20850 USA 301 762 0888 866 360 9531 inquiry genecopoeia com www genecopoeia com 2009 GeneCopoeia Inc All in One First Strand CDNA Synthesis Kit USER MANUAL I All in One First Strand cDNA Synthesis Kit l Description Il Related Products Ill Contents and Storage IV Preparation V Procedure VI Example VII Trouble Shooting Guide VIII Limited Use License and Warranty I Description The All in One First Strand cDNA Synthesis Kit includes a reverse transcriptase and a specialized set of reagents designed to yield first strand cDNA that is optimal for gene cloning cDNA library creation and quantitative PCR amplification A robust experimental design delivers a universal kit that is suitable for first strand cDNA synthesis from almost any source of RNA The kit uses Moloney Murine Leukemia Virus Reverse Transcriptase RNase H Minus M MLV RTase H which is an RNA dependent DNA polymerase used in cDNA synthesis with long RNA templates gt 13kb The lack of RNase H activity is important in this application in that RNase H activity will start to degrade template during long incubation times requir2. The wrong product was amplified Optimize the PCR reaction conditions All in One First Strand CDNA Synthesis Kit Vill Limited Use License and Warranty Limited Use License Following terms and conditions apply to use of all OmicsLink ORF Expression Clones in all lentiviral vectors and Packaging Kit the Product If the terms and conditions are not acceptable the Product in its entirety must be returned to GeneCopoeia 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 GeneCopoeia 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 GeneCopoeia warrants that the Product meets the specifications described in the accompanying Product Datasheet If it is proven to the satisfaction of GeneCopoeia that the Product fails to meet these specifications GeneCopoeia will replace the Product In the event a replacement cannot be provided GeneCopoeia will provide the purchaser with a refund This limited warranty sha
3. e The final volume should be 13ul Reagents Volume Final concentration Total RNA 1 ug or polyA RNA or 10 ng t 250 uM Random Primer 10 uM or 60 uM Oligo dT js 1 yl or 2 4uM or 10 uM sequence specific primer or 0 4 uM tt ddH20 RNase DNase free to 13 ul t The amount of RNA in the table is the recommended amount The total RNA may be adjusted to between 10 ng 5 yg and the purified poly A RNA between 1 ng 100 ng tt Please choose one of the RT Primers based on the experimental design The reverse transcription will begin at the polyA tail if using the Oligo dTs It will begin at many different RT sites throughout the RNA if using the Random Primer All in One First Strand CDNA Synthesis Kit Denature RNA Mix the reaction solution well Spin down briefly Heat the RNA Primer mix at 65 C for 10 minutes then cool it down immediately on ice Prepare RNA reverse transcription reaction Add the following reagents into the RNA Primer mix reaction tube which has been cooled on ice The final volume should be 25 ul Reagents Volume Final concentration RNA Primer Mix 13 ul 5X RT Reaction Buffer 5 ul 1X 25 mM dNTP 1 ul 1 mM 25 U l RNase Inhibitor 1 ul 1U 200 U ul M MLV RTase 1 ul 8U ddH20 RNase DNase free to 25 ul Reverse Transcription Reaction Mix reaction solution well Spin down briefly Incubate the reaction solution at 37 C for 60 minutes if using the Rand
4. ed for producing long cDNAs RNase H minus RT enables preparation of long cDNAs and libraries containing a high percentage of full length cDNA ll Related Products GeneCopoeia Description All in One qPCR Mix SYBR Green based real time quantitative PCR Mix All in One qPCR Primers Human mouse and rat primers Validated gene specific primers ensure specificity and sensitivity All in One qPCR Primer Array User specified ready to use primer arrays in 96 well plate format Reliable tools ideal for analyzing the expression of a focused panel of genes such as pathways diseases or customized gene panels All in One miRNA gRT PCR Detection Kits SYBR Green based Accurately quantify miRNA expression All in One miRNA qPCR Primers Human mouse and rat primers Validated for robust reproducible and reliable quantitation of miRNA activity OmicsLink Expression Ready ORF cDNA Clones 20 000 human 15 000 mouse Perform a variety of applications with expression ready clones Endofectin Transfection Reagents Optimized for specific cell types Transfect efficiently and with low toxicity All in One First Strand CDNA Synthesis Kit lll Contents and Storage Contents and storage recommendations for the All in One First Strand cDNA Synthesis Kit Cat Nos AORT 0020 and AORT 0050 are provided in the following table Contents Quantity S
5. ll not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to GeneCopoeia within 30 days of receipt of the Product GeneCopoeia s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price GeneCopoeia s liability does 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 GeneCopoeia does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose GeneCopoeia is committed to providing our customers with high quality products If you should have any questions or concerns about any GeneCopoeia products please contact us at 301 762 0888 2009 GeneCopoeia Inc GeneCopoeia Inc 9620 Medical Center Drive Rockville MD 20850 Tel 301 762 0888 Fax 301 762 3888 Email inquiry genecopoeia com Web www genecopoeia com GeneCopoeia Products are for Research Use Only Copyright 2010 GeneCopoeia Inc Trademarks GeneCopoeia OmicsLink All in One EndoFectin GeneCopoeia Inc SYBR Molecular Probes iQ 5 Bio Rad ROX Invitrogen AOFS1 0210
6. oggles 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 250 C for 3 hours Use DEPC to treat all microcentrifuge tubes pipettes and pipette tips if not RNase free and then autoclave to deactivate RNases RNase free consumables 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 Mix reagents thoroughly by gently inverting tubes several times avoiding bubbles and then briefly centrifuge before use 3 Setup all reactions on ice to reduce risk of RNA degradation 4 Read all procedures before setting up RT reaction V Procedure 1 Thaw all the reagents needed for RNA reverse transcription from the First Stand cDNA Synthesis Kit Mix reagents well by gently inverting the tubes Spin down briefly and keep on ice 2 Prepare the RNA Primer Mix Add the following reagents into an RNase free reaction tube which has been pre cooled on ic
7. om Primer or incubate at 42 C for 60 minutes if using the oligo dT 1g or sequence specific primer Terminate the reaction by heating at 85 C for 5 minutes and then store at 20 C The cDNA reaction product can be used directly in the next step without being purified A volume of 0 5 ul 2 ul of undiluted cDNA is recommended for standard 25 ul PCR reactions If performing quantitative PCR it is recommended to do a 1 5 1 20 dilution of the cDNA and add a volume of 2 ul for each 20 ul qPCR reaction All in One First Strand CDNA Synthesis Kit VI Example Objective The reverse transcription efficiency of the All in One First Strand Synthesis Kit is assessed by examining the amplification results of different genes or gene regions using the oligo dT synthesized cDNA prepared from the All in One First Strand cDNA Kit M12 3 45 6 7 89 1011121314 Lane symbol Amp Region PCR Size 1 2 USPS9Y 1 59 1 63kb 142bp 3 4 SENP5 3 84 3 99kb 151bp 5 6 MTHFR 5 21 5 34kb 130bp 7 8 PRKCA 7 06 7 20kb 139bp 9 10 FBXW2 7 82 7 96kb 141bp 11 12 ENTPD1 11 0 11 15kb 154bp 13 14 MACF1 12 92 13 01kb 132bp M 100bp Plus DNA Ladder Cat M01010A RN A template 5 3 3 L meal ene nec TR sees o use O 5 415kb 413kb 411kb Skb kb kb 3kb 41kb PCR Amp Region Synthesis cDNA Oligo dT Figure 1 Efficient cDNA synthesis by All in One First Strand cDNA Synthesis Kit Total RNA isolated from human placenta was used as template RNA in reverse
8. torage temperature conditions 200 U ul M MLV Reverse Transcriptase RNase H 1x 20 ul 1x 50 ul 20 C Stable for at least 12 months Alternatively the solution can also be stored at 80 C in aliquots Avoid repeated freezing thawing 5X RT Reaction Buffer 1x 100 ul 1 x 250 ul 20 C Stable for at least 12 months Alternatively the solution can also be stored at 80 C in aliquots Avoid repeated freezing thawing 25 U ul RNase Inhibitor 20 C Stable for at least 12 months Alternatively the solution can also be stored at 80 C in aliquots Avoid repeated freezing thawing 25 mM dNTP 20 C Stable for at least 12 months Alternatively the solution can also be stored at 80 C in aliquots Avoid repeated freezing thawing 60 uM Oligo dT 20 C Stable for at least 12 months Alternatively the solution can also be stored at 80 C in aliquots Avoid repeated freezing thawing 250 uM Random Primer 20 C Stable for at least 12 months Alternatively the solution can also be stored at 80 C in aliquots Avoid repeated freezing thawing dd H20 RNase and DNase free 20 C Stable for at least 12 months Alternatively the solution can also be stored at 80 C in aliquots Avoid repeated freezing thawing All in One First Strand CDNA Synthesis Kit IV Preparation Wearing a lab coat disposable gloves and protective g
9. transcription reactions using the All in One First Strand cDNA Synthesis Kit together with the oligo dT primer The synthesized cDNA was then used to amplify different gene regions by quantitative PCR using the All in One qPCR Mix GeneCopoeia Catalog No AOPR 0200 The positive amplification results of MACF1 indicate that up to a 13 kb RNA sequence was reversed transcribed All in One First Strand CDNA Synthesis Kit Vil Trouble Shooting Guide Little or no RT PCR product RNA template degradation The quality of the RNA is the key factor for cDNA synthesis Follow the RNA isolation kit procedure carefully always wearing a lab coat gloves and mask when 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 template 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 digest before the RT step or design intron spanning or flanking primers to avoid co amplification of genomic DNA
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