All-in-One - GeneCopoeia
Contents
1. Do not add more than 5 of the original cDNA solution volume to the total qPCR reaction solution ROX Reference Dye is added only for qPCR instruments that require ROX for calibration ROX Reference Dye provides an internal reference to which the reporter dye signal can be normalized during data analysis Normalization is necessary to correct for fluorescence fluctuations due to changes in concentration or volume Adjust the ROX Reference Dye to optimal concentration according to different qPCR instruments All in One qPCR Mix Manual Instrument ROX per 20ul PCR Reaction Final Concentration BioRad iCycler MyiQ iQ5 CFX 96 CFX 384 Eppendorf Mastercycler realplex Roche LightCycler 480 LightCycler 2 0 None No ROX ABI PRISM 7000 7300 7700 7900HT and 7900HTFast ABI Step One ABI Step One Plus 0 4 ul 0 2 0 4u1 600 nM 300 600nM ABI 7500 7500 Fast ABI Viia7 Stratagene Mx3000P Mx3005P Mx4000 0 1 ul 0 02 0 1 pI 150 nM 30 150nM For other instruments which need calibration of ROX but have not been listed out in the table please optimize the concentration of ROX according to the guide line of specific instrument Mix the PCR reaction mix sufficiently and add to the PCR reaction tubes Briefly centrifuge to make sure all the reagents are at the bottom of the reaction tubes The following three step method for programming the PCR reac2. 1x PCR forward primer 2 uM 0 2 uM PCR reverse primer 2 uM 0 2 uM ROX Reference Dye 30uM if needed Water double distilled 600nM 150nM a Not using ROX Reference Dye Using ROX Reference Dye Total Use the 2xAll in One qPCR 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 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 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 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
3. 2x1 ml Alternatively the solution can also be 5x 2x1 ml stored at 80 C in aliquots Avoid repeated freezing thawing 2xAll in One qPCR Mix 20x 2x1 ml 1x80 ji 20 C Stable for at least 12 months POAREIRERGE DYE 3x80 p Alternatively the solution can also be 30M 5x80 ul stored at 80 C in aliquots Avoid 20x80 ul repeated freezing thawing IV Preparation Wearing a lab coat disposable gloves and protective goggles are recommended when handling chemicals IMPORTANT NOTES When using the All One qPCR Mix with miProfile miRNA qPCR Arrays and All in One miRNA First Strand cDNA Synthesis Kit for miRNA expression profiling please follow the miProfile miRNA qPCR array user manual for the complete instruction 2 Store the kit at 20 C Avoid storage or leaving reagents at 4 C or room temperature Avoid light exposure at all times 3 Mix reagents thoroughly by gently inverting tubes several times avoiding bubbles and then briefly centrifuge before use 4 Prepare the reaction mix with PCR grade water 5 Strictly follow standard procedures for PCR to avoid nucleic acid contamination and non specific amplification 6 Read all procedures before setting up the PCR reaction V Procedure 1 Thaw the 2xAll in One qPCR Mix and ROX Reference Dye as needed 2 Prepare the PCR reaction mix on ice See the example below All in One qPCR Mix Manual 2xAll in One qPCR Mix 10 ul
4. 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 2014 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 2014 GeneCopoeia Inc Trademarks GeneCopoeia All in One ExProfile miProfile GeneCopoeia Inc RNAzol Molecular Research Center Inc SYBR Molecular Probes iQ 5 Bio Rad ROX Invitrogen AOPR 081414 10
5. 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 1000 1000 qPCR reactions Cat No AOPR 4000 4000 qPCR reactions Performance optimized for All In One qPCR Primers All In One miRNA qPCR Primers miProfile miRNA qPCR Arrays ExProfile Gene qPCR Arrays All In One First Strand cDNA Synthesis Kit and All In One miRNA First Strand cDNA Synthesis Kit 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 2014 GeneCopoeia Inc All in One qPCR Mix Manual USER MANUAL All in One qPCR Mix Description Il Related Products lll 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 qPCR 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 or miRNAs 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 Il Related P
6. another commercial source is used please reference the instrument manual and adjust the extention time and melting curve conditions accordingly 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 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 l 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 N o Ne oar Heating Rate Melting curve reading 72 C 95 C 05 C 6 sec Yes T 2500 a A 8 8 8 8 PCR Base Line Subtracted Curve Fit RFU 8 All in One qPCR Mix Manual 6 Analyze the amplification and corresponding melting curves after the qPCR experiment a Amplification curves of serially d
7. e 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 sample 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 Abnormal melting curves Signal in the blank No Template Conirol sample 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 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 does not work redesign the primers Double peaks and multiple peaks in the melting curve of the positive control In the absence of other
8. iluted plasmid DNA Peak values of amplified products in melting curves 7 Construct a standard curve using the Ct values from each amplification curve 35 30 25 Threshold Cycle 20 is T T T T T T T T T 2 Log Starting Quantity copy number 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 All in One qPCR Mix Manual VII Trouble Shooting Guide Poor precision or failed qPCR reactions Make sure the initial denature time was set as 10min sufficiently activating of the hot start polymerase could avoid non specific amplification and production of primer dimers 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 th
9. ply 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 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
10. 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 8 All in One qPCR Mix Manual Prepare the 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 No peaks or abnormal peaks in the melting curve or the amplification curves of the positive control e Adjust the ROX Dye to optimized concentration according to instrument 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 ap
11. roducts GeneCopoeia offers comprehensive solutions for studying gene expression A careful process of co development ensures that they work well together and provide robust and reproducible results Product Description All in One First Strand cDNA Synthesis Kit Reverse transcribe mRNA into first stand cDNA All in One qPCR Primers Validated gene specific primers ensure specificity and sensitivity human mouse and rat ExProfile Gene qPCR Arrays High throughput or focused group profiling of gene expression All in One miRNA First Strand cDNA Synthesis Kit Reverse transcribe miRNA into first stand cDNA All in One miRNA qRT PCR Detection Kits SYBR Green based detection kit accurately quantifies miRNA expression All in One miRNA gPCR Primers Validated human mouse rat miRNA primers for robust reproducible and reliable quantitation of miRNA activity miProfile miRNA gPCR Arrays High throughput or focused group profiling of miRNA expression RNAzol RT RNA Isolation Reagent Easy isolation of mRNA microRNA or total RNA All in One qPCR Mix Manual lll Contents and Storage Contents and storage recommendations for the All in One qPCR Mix are provided in the following table Cat Nos AOPR 0200 AOPR 0600 AOPR 1000 and AOPR 4000 Conients Quantity Storage temperature conditions 2x1 ml ont 20 C Stable for at least 12 months 3x
12. tion is recommended 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 15 sec Yes Notes i When using SYBR Green dye to monitor the qPCR reaction a melting curve analysis should be performed immediately at the end of cycling example adapted from the iQ5 real time PCR detection system from Bio Rad Temperature range Heating rate Constant temperature Detection 72 95 C 0 5 C unit time 6 sec unit time Yes 25 C 30 sec No The conditions for your instrument may differ for instructions consult the documentation of your qPCR instrument All in One qPCR Mix Manual ii The DNA polymerase used in the 2xAll in One qPCR Mix is a special chemically modified hot start enzyme Incubation for 10 minutes at 95 C will sufficiently activate the enzyme iii 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 iv The optimal fragment length to use for amplification during real time PCR is in the range of 80 150bp However fragment lengths up to 300bp are possible v The main condition for the above reaction are referred to in the iQ5 qPCR instrument manual from Bio Rad If a qPCR instrument from
Download Pdf Manuals
Related Search