All-in-One™ first-strand cDNA synthesis kit user manual
Contents
1. e Genomic DNA was present Perform a DNase digest before the RT step or design intron spanning or flanking primers to avoid co PCR prod ct is longer amplification of genomic DNA than expected e The wrong product was amplified Optimize the PCR reaction conditions All in One First Strand CDNA Synthesis Kit VIII Limited Use License and Warranty Limited Use License nn Following terms and conditions apply to use of all All in One First Strand cDNA Synthesis Kits 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 sp2. 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 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 VII Trouble Shooting Guide RNA template degradation e 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 Little or no RT PCR An inhibitor was present in the RNA template product e 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 e Prepare the RNA Primer Mix before the RT step Then add a PCR enhancing reagent such as DMSO betaine etc in the PCR reaction
3. 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 labvvare 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 Set up 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
4. Mix as half of the total reaction volume and adjust other reagents accordingly If the total reaction volume is changed maintain each component in the proper proportion b Primers are important considerations to ensure success with real time PCR All in One human mouse and rat primer sets from GeneCopoeia have been validated to provide specific and sensitive amplification even with low copy number genes For designing your own primers you may wish to use Oligo primer analysis software Molecular Biology Insights or Primer Premier software Premier Biosoft International c Primer concentration should be in the range of 0 2 to 0 6 uM In general a PCR reaction using 0 2 uM primers produces good results If the PCR efficiency is low consider increasing primer concentration However keep in mind that non specific PCR products may also increase with increased primer concentration d Generally the amount of DNA template should be less than 100 ng Because different templates contain varying copies of a target gene it may be necessary to perform a gradient dilution to determine the optimal amount of DNA template to use If reverse transcript cDNA is used as template dilute before use Do not add more than 5 of the original cDNA solution volume to the total GPCR reaction solution e ROX Reference Dye is added only for qPCR instruments that require ROX for calibration 3 Mix the PCR reaction mix sufficiently and add to the PCR reaction tu
5. of co develop ment ensures that they work well together and provide robust and reproducible results GeneCopoeia Description All in One First Strand cDNA Synthesis Kit Reverse transcription kit Produces first strand cDNA using poly A or total RNA as template All in One qPCR Primers Human mouse and rat primers Validated gene specific primers ensure specificity and sensitivity All in One miRNA qRT 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 miProfile miRNA qPCR Arrays User specified ready to use primer arrays in 96 or 384 well plate Reliable tools ideal for analyzing the expression of a focused panel of genes such as pathways diseases or customized gene panels 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 qPCR Mix Manual lll Contents and Storage Contents and storage recommendations for the All in One qPCR Mix Cat Nos AOPR 0200 AOPR 0600 AOPR 1200 AOPR 1000 AOPR 4000 are provided in the following table Contents Quantity Stor
6. tube which has been pre cooled on ice The final volume should be 13ul Reagents Volume Final concentration Total RNA 1 ug or polyA RNA or 10 ng 250 uM Random Primer 10 uM or 60 uM Oligo dT 18 1ul or 2 4uM or 10 uM sequence specific primer or 0 4 uM t Spike in control RNA Only comes in All in One first strand cDNA Tul synthesis kits ddH2O RNase DNase free to 13 ul All in One First Strand CDNA Synthesis Kit t The amount of RNA in the table is the recommended amount The total RNA may be adjusted to between 10 ng 5 ug 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 dT 1g It will begin at many different RT sites throughout the RNA if using the Random Primer ttt Spike in control RNA is used to monitor the efficiency of the RT reactions 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 1mM 25 U ul RNase Inhibitor 1 ul 1U 200 U ul M MLV R
7. 0 sec No VI Example Objective The amplification efficiency and detection sensitivity of the 2xAll in One qPCR Mix are assessed by standard curves made by gradient dilution of plasmid DNA The target fragment is 102 bp Equipment iQ5 instrument Bio Rad Laboratories Procedure 1 The plasmid is serially diluted to 6 concentrations ranging from 10 to 1 molecule l 2 PCR reaction mix preparation on ice All in One qPCR Mix Manual Reagent components Volume 2xAll in One qPCR Mix 10 ul PCR forward primer 2 uM 2 ul PCR reverse primer 2 UM 2 ul ddH20 1 ul Total 15 ul 3 Mix the above reagents sufficiently Aliquot to PCR tubes after a brief centrifugation 4 Add 5 ul of the diluted plasmid template to each PCR tube Use 5ul ddH2O as a negative control 5 Program the PCR reaction and corresponding reading conditions of the melting curve 1 Initial denaturation 10 min No I PaB Heating Rate Melting curve reading 72 C 95 C 0 5 C 10 sec Yes 6 Analyze the amplification and corresponding melting curves after the qPCR experiment 1 o F i E Hi SYER ee ee es ee O E S 10 15 2 5 x 35 0 45 75 El se ee ee EE E ad Amplification curves of serially diluted plasmid DNA Peak values of amplified products in melting curves All in One qPCR Mix Manual 7
8. Construct a standard curve using the Ct values from each amplification curve 35 30 _ 2 es LE e 3 25 2 a 5 ms sn lt Sx 20 15 T T T T T T T T T T T T o 2 4 6 Log Starting Quantity copy number e SYBR E 99 9 RA2Z 1 000 slope 3 324 y int 34 872 Picture of a standard curve 8 Conclusion The peak values from the amplification and melting curves show that as low as 5 molecules can be detected when using plasmid DNA as a template and that there is only a single amplified product showing that very high sensitivity can be attained using the All in One qPCR Mix At the same time high amplification efficiency is also shown by the good linear relationship among each concentration of serially diluted plasmid VII Trouble Shooting Guide Poor precision or failed qPCR reactions The fluorescence detection temperature may not be appropriate Adjust accordingly The set up position for reaction samples in the real time PCR instrument may not be right Adjust accordingly PCR cycle conditions primer concentration and primer sequences may not be appropriate Adjust the primer concentration and annealing temperature If this does not work redesign the primers The template sample purity may not be adequate Purify the template sample by phenol chloroform extraction and ethanol precipitation If the samples are reverse transcribed cDNA set up the qPCR reaction with a diluted sam
9. 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 0060 60 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 2013 GeneCopoeia Inc All in One First Strand CDNA Synthesis Kit USER MANUAL All in One First Strand cDNA Synthesis Kit 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 requir
10. Tase 1 ul 8U ddH2O 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 Random Primer or incubate at 42 C for 60 minutes if using the oligo dT ig 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 8 9 1011121314 Lane symbol Amp Region PCR Size 1 2 USP9Y 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 FBXVW2 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 Catt M010104 RNA template 5 3 3r IT TS DT NN NT DT sey ue 1516 143kb 114kb Si 7kb 5kb 3kb 1kb
11. age temperature conditions 2x1 ml 3x 2x1 ml 20 C Stable for at least 12 months 2xAll in One qPCR Mix 6x 2x1 ml Alternatively the solution can also be stored at 80 C in aliquots Avoid 1x12 ml repeated freezing thawing 4x 1x12 ml 1x80 pl 3x80 pl 20 C Stable for at least 12 months 50xROX Reference Dye 6x80 pl Alternatively the solution can also be stored at 80 C in aliquots Avoid 1x450 pl repeated freezing thawing 4x 1x450 ul IV Preparation Wearing a lab coat disposable gloves and protective goggles are recommended when handling chemicals IMPORTANT NOTES 1 Store the kit at 20 C Avoid storage or leaving reagents at 4 C or room temperature Avoid light exposure at all times 2 Mix reagents thoroughly by gently inverting tubes several times avoiding bubbles and then briefly centrifuge before use 3 Prepare the reaction mix with PCR grade water 4 Strictly follow standard procedures for PCR to avoid nucleic acid contamination and non specific amplification 5 Read all procedures before setting up the PCR reaction V Procedure 1 Thaw the 2xAll in One qPCR Mix and 50xROX Reference Dye as needed 2 Prepare the PCR reaction mix on ice See the example below All in One qPCR Mix Manual Reagent Volume per reaction Final concentration en Ee Ed ee es a Dl 50xRox Reference Dye 0 4 ul x Water double distilled Po a Use the 2xAll in One qPCR
12. also be stored at 80 C Transcriptase RNase H 3x20 ul in aliquots Avoid repeated freezing thawing 1x 100 ul 20 C Stable for at least 12 months 5X RT Reaction Buffer Alternatively the solution can also be stored at 80 C 3x 100 ul in aliquots Avoid repeated freezing thawing 20 C Stable for at least 12 months 25 U ul RNase Inhibitor Alternatively the solution can also be stored at 80 C in aliquots Avoid repeated freezing thawing 20 C Stable for at least 12 months 25 mM dNTP Alternatively the solution can also be stored at 80 C in aliquots Avoid repeated freezing thawing 20 C Stable for at least 12 months 60 uM Oligo dT 18 Alternatively the solution can also be stored at 80 C in aliquots Avoid repeated freezing thawing 20 C Stable for at least 12 months 250 uM Random Primer Alternatively the solution can also be stored at 80 C in aliquots Avoid repeated freezing thawing 20 C Stable for at least 12 months Spike in control RNA for use Alternatively the solution can also be stored at 80 C with gene qPCR array only in aliquots Avoid repeated freezing thawing 20 C Stable for at least 12 months dd H2O RNase and DNase Alternatively the solution can also be stored at 80 C free in aliquots Avoid repeated freezing thawing All in One First Strand CDNA Synthesis Kit IV Preparation Wearing a lab coat
13. bes 4 Briefly centrifuge to make sure all the reagents are at the bottom of the reaction tubes 5 The following three step method for programming the PCR reaction is recommended All in One qPCR Mix Manual Cycles Steps Temperature Time Detection 1 Initial denaturation 95 C 10 min No Denaturation 95 C 10 sec No 40 Annealing 55 C 60 C 20 sec No Extension 72 C At least 15 sec Yes a The DNA polymerase used in the 2xAll in One qPCR Mix is a special chemically modified hot start enzyme The indicted initial denaturation is sufficient to activate the enzyme b The actual annealing temperature should be adjusted around the primer melting temperature ranging from 55 C 60 C However the optimal annealing temperature may be outside of this range Adjust the temperature according to actual reaction conditions c The extension time is specific for the instrument See the documentation provided with your instrument d When using SYBR Green dye to monitor the qPCR reaction a melting curve analysis should be performed immediately after qPCR cycling For instructions consult the documentation for your qPCR instrument The following is an example adapted from the iQ5 real time detection system from Bio Rad Laboratories The conditions for your instrument may differ Temperature range Heating rate Constant temperature Detection 72 95 C 0 5 C unit time 10 sec unit time Yes 25 C 3
14. e qPCR reaction mix on ice optimize the qPCR reaction conditions for example by increasing the annealing temperature decreasing the primer concentration or increasing the fluorescence detection temperature not more than the Tm value of the expected product If this does not work redesign the forward primer e Not enough PCR cycles For good sensitivity one should generally set up more than 35 PCR cycles but more than 45 cycles may result in too much background signal e The amount of template used may not be enough or the template may No signal Ct or late be degraded Use the highest concentration possible of diluted appearing signal template samples to set up the qPCR At the same time avoid freezing and thawing the samples repeatedly e The amplification efficiency is low and the qPCR reaction conditions are not optimal Redesign the primers and optimize the reaction conditions All in One qPCR Mix Manual VIII 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 no
15. ecifications GeneCopoeia will replace the Product In the event a replacement cannot be provided GeneCopoeia 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 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 2013 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 2013 GeneCopoeia Inc Trademarks GeneCopoeia OmicsLink All in One ExProfile miProfile EndoFectin GeneCopoeia Inc SYBR Molecu
16. 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 Product Description All in One qPCR Mix SYBR Green based real time quantitative PCR Mix All in One qPCR Primers Validated gene specific primers ensure specificity and sensitivity RNAzol RT RNA Isolation Reagent Easy isolation of MRNA microRNA and total RNA ExProfile Gene qPCR Arrays For expression profiling of pre defined or customized sets of genes in various tissues or cells All in One miRNA qRT PCR Detection Kits Accurately quantify miRNA expression All in One miRNA qPCR Primers Validated for robust reproducible and reliable quantitation of miRNA activity miProfile miRNA qPCR Arrays For expression profiling of pre defined or customized sets of miRNAs in various tissues or cells OmicsLink Expression Ready ORF cDNA Clones Perform a variety of applications with expression ready clones 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 0060 are provided in the following table Contents Quantity Storage temperature conditions 1x20 ul 20 C Stable for at least 12 months 200 U ul M MLV Reverse Alternatively the solution can
17. lar Probes iQ 5 Bio Rad ROX Invitrogen RNAzol Molecular Research Center Inc AOFS1 062813 8 Expressway to Discovery All in One qPCR Mix For universal quantitative real time PCR Cat No AOPR 0200 200 qPCR reactions Cat No AOPR 0600 600 qPCR reactions Cat No AOPR 1200 1200 qPCR reactions Cat No AOPR 1000 1000 qPCR reactions Cat No AOPR 4000 4000 qPCR 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 2012 GeneCopoeia Inc All in One qPCR Mix Manual USER MANUAL I All in One qPCR Mix 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 gPCR Mix provides fast and efficient SYBR Green based real time quantitative PCR The qPCR Mix uses a high fidelity hot start DNA polymerase optimized reaction buffer and high quality dNTPs to enable specific and sensitive amplification of even low copy genes The All in One qPCR Mix reduces experimental design time by providing a universal reaction condition that can be used with almost all primers and most real time PCR instruments ll Related Products GeneCopoeia offers comprehensive solutions for studying gene expression A careful process
18. ple as other concentrated reagents in the RT reaction mixture may be interfering with the qPCR Try to use 3 0 agarose gel electrophoresis to check the qPCR products Check the purity of the primers by electrophoresis or use PAGE purified primers if the bands are diffused One may also use phenol chloroform extraction and ethanol precipitation methods to treat the primers before the experiment All in One qPCR Mix Manual Signal in the blank No Template Control sample e There may be contamination of the positive samples in the qPCR reaction system if the Tm of the melting curve of the blank control is the same as the positive control Eliminate sample application error first If the situation still persists replace the PCR grade water and or primers and or use a new 2xAll in One qPCR Mix e If the Tm of the melting curve of the blank control is lower than the positive control the qPCR reaction may have produced nonspecific amplification such as primer dimers Prepare the qPCR reaction mix on ice and increase the temperature of fluorescence detection If this Abnormal melting does not work redesign the primers curves Double peaks and multiple peaks in the melting curve of the positive control e In the absence of other primers present in the reaction double or multiple peaks in the melting curve of the positive control indicate that the qPCR reaction produced nonspecific amplification fragments Prepare th
19. t 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 shall 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 merchantabili
20. ty 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 101 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 2012 GeneCopoeia Inc Trademarks GeneCopoeia OmicsLink All in One EndoFectin GeneCopoeia Inc SYBR Molecular Probes iQ 5 Bio Rad ROX Invitrogen AOPR 041013
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