User`s Manual
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1. knockdown verification e Array Validation Bio Chain Institute Inc Website www biochain com t 888 762 2568 f 510 783 5386 e mail info biochain com QCell Pro One Step qRT PCR SuperMix Kit 020107V2 Description Components in this kit are prepared with pure chemicals according to our proprietary technology QCell Pro One Step qRT PCR SuperMix Kit provides a one step simple robust inexpensive assay for detection and quantitative analysis of gene expression directly from cells or RNA with probe based format Quality Control 1 kit of this lot has been tested for quantitating human GAPDH gene expression in a serial dilution of cell lysate from 216 cells to 1 cell using Stratagene s Mx3005P as a real time PCR instrument Good linearity and great PCR efficiency is observed and consistent with the previous lot Components Catalog Number K5055200 Reagents are sufficient for 200 assays item mont Fart NO K5056200 1 Pro QRT PCR Reaction Mixture 2x containing 20 M X K50 00 Hotstart Taq DNA polymerase 3 Reverse Transcriptase RNase Inhibitor Mixture 100 ul K5055200 3 4 ROX Reference Dye 50 ul x2 K5055200 4 5 Nuclease Free PCR Grade Water K5055200 5 Catalog Number K5055400 Reagents are sufficient for 400 assays WMI 2 2 ea eS PartNo K5055400 1 2 Pro qRT PCR Reaction Mixture 2x containing 1 25 ml x4 K5055400 2 Hotstart Taq DNA Polymerase 3 Reverse Transcriptase RNase Inhibitor Mixture 100 u2. 1 Harvest cells using the method appropriate to the properties of the cell line For adherent cells trypsinize the cells using standard techniques Count the cell 2 Pelleting the cells by centrifuging at 200 300x g for 5 min Carefully remove the supernatant by aspiration 3 Wash the pellet once with ice cold PBS Pelleting the cells by centrifuging at 200 300x g for 5 min Carefully remove the supernatant by aspiration Keep the pellet on ice 4 Add appropriate volume of Cell Lysis Buffer to the cell pellet Vortexing for 1 minute to lyse the cells 5 Analyze the lysate by RT PCR RNAs in the lysate are stable at 4 C for up to 4 hr QRT PCR setup and cycling 1 Prepare the following RT PCR reaction mixture First make the master mix without the template After making the master mix gently mix the reaction without creating bubbles aliquot and then add 1 2 5 ul of template to each experimental reaction per reaction 25 ul inal Concentration x Reverse Transcriptase RNase 0 5 ul inwotortintwe 150 200 nM X 150 200 nM 150 500 nM ROX Reference Dye Template cell lysate or RNA 1 2 5 ul Nuclease free PCR grade water Add up to 25 ul See page 4 Use of the ROX Reference Dye If cell lysate is used as the template the volume of cell lysate should not exceed 1 10 volume of the qRT PCR reaction If RNA is used as the template it is recommended to use RNA template in less than 1 ug 2 Gentl
3. Higuchi R Fockler C Dollinger G and Watson R 1993 Kinetic PCR analysis real time monitoring of DNA amplification reactions BioTechnology 11 1026 1030 3 Bustin S A 2000 Absolute quantification of mRNA using real time reverse transcription polymerase chain reaction assays Journal of Molecular Endocrinology 25 169 193 5 Bio Chain Institute Inc Website www biochain com t 888 762 2568 f 510 783 5386 e mail info biochain com
4. QCell Pro One Step qRT PCR SuperMix Kit 020107V2 User s Manual and Instructions Product QCell Pro One Step qRT PCR SuperMix Kit Catalog Number K5055200 K5055400 Introduction qRT PCR is a highly sensitive technique that is widely used for detection and quantification of RNA in tissues and cultured cells Traditionally quantitative PCR is performed in two steps a first strand cDNA synthesis step using reverse transcriptase followed by a PCR step using a thermostable DNA polymerase This Kit combines Reverse Transcriptase MMLV RTase and RNase Inhibitor in a single mixture with hotstart Taq DNA polymerase in a separate 2x reaction mix optimized for probe based qRT PCR Both cDNA synthesis and PCR are performed ina single tube using gene specific primers and either cell lysate or RNA A cell lysis buffer is provided in the kit to make cell lysates in less than 5 minutes at room temperature The cell lysate can be used directly for RT PCR bypassing RNA isolation procedure The passive reference dye ROX is included in a separate tube to make the QCell Pro One Step qRT PCR SuperMix adaptable for many real time QPCR platforms BioChain s QRT PCR SuperMix contains BioChain s Taq polymerase with hot start capability BioChain s hot start Taq polymerase improves PCR amplification reactions by decreasing non specific amplification and preventing primer dimer formation This enzyme is activated after an initial 10 minutes heating at 95 C An
5. d the real time RT PCR buffer is specially formulated to provide superior specificity and increase reverse transcription and amplification efficiency BioChain Institute Inc Website www biochain com t 888 762 2568 f 510 783 5386 e mail info biochain com QCell Pro One Step qRT PCR SuperMix Kit 020107V2 Fluorescence dRn es 2 4 6 3 10 12 14 16 13 20 22 24 26 28 30 32 34 36 38 40 Cycles Y 3 304Log x 31 88 R 0 999 Efficiency 100 8 o u o WN 0 we j Ct aRn 53 os 26 100 Initial Quantity Cell number Figure 1 BioChain s QCell Pro One Step QRT PCR SuperMix provides sensitive detection down to a single cell K562 cells were lysed according to the cell lysis protocol 6 fold serial dilution of cell Isyate were prepared from 216 cells to 1 cell GAPDH gene expression was detected using BioChain s QCell Pro qRT PCR kit on Stratagene s Mx3005P instrument Efficiency as measured from standard curve was 100 8 with a R value of 0 999 Features e Flexible and convenient quantitating gene expression in cells without isolating RNA or RNA in one step format e Save time quick cell lysis procedure and ready to use supermix reducing setup time and liquid handling steps e High Sensitivity qRT PCR from as low as 1 cell or 1 pg total RNA e Versatile compatible with a wide variety of cell lines Applications e Real Time RT PCR e Gene expression profiling e Gene
6. ion keep all solutions containing the ROX protected from light 2 Due to the sensitivity of quantitative PCR results can be easily affected by pipetting errors Always prepare a master mix of qRT PCR supermix containing the primers and the reference dye if reference dye is used Individual pipetting of replicate samples is not recommended BioChain Institute Inc Website www biochain com t 888 762 2568 f 510 783 5386 e mail info biochain com QCell Pro One Step qRT PCR SuperMix Kit 020107V2 Cell Lysis Procedure The lysis buffer can be used to prepare lysates from a vari ety of mammalian culture cells Lysates may be prepared with the maximum cell density 10 cells ul When used for qRT PCR the lysate may be diluted in the cell lysis buffer prior to adding to the qRT PCR reaction High concentration of either cellular materials or lysis buffer may inhibit qRT PCR reaction so the total amount of cell lysate added to the qRT PCR reaction should not exceed 1 10 volume of the reaction And the number of cells added to the 25 ul qRT PCR reaction should be lt 2 000 This is a general guideline For some cells lines 2 000 cells may inhibit the qRT PCR reaction Prior to the experiment perform a pilot standard curve to determine the maximum number of the cells that may be added to the qRT PCR reaction and determine the cell number range that give linear amplification of the specific target under your specific reaction conditions
7. l x2 K5055400 3 S0nIx4 KS056400 4 5 Nuclease Free PCR Grade Water K5055400 5 BioChain Institute Inc Website www biochain com t 888 762 2568 f 510 783 5386 e mail info biochain com QCell Pro One Step qRT PCR SuperMix Kit 020107V2 Reagents and Equipments Required but not Supplied in this Kit 1 PBS Ca Mg free 2 Spectrofluorometric thermal cycler Storage and Stability Upon receipt store all components at 20 C in a constant temperature freezer Avoid repeated freeze thaw cycles When stored under these conditions the supermix is stable for one year after ship date The ROX reference dye is light sensitive and should be kept away from light whenever possible Protocol Primer and Probe Design Design QPCR primers to generate amplicons of lt 150 bp Since the cell lysate contains genomic DNA the primers and probe should be designed to amplify cDNA but minimize amplification of genomic DNA It is useful to choose primers or probe that span an exon exon boundary in the target mRNA or choose primers that flank a large intron If possible design primers and probe to avoid regions of secondary structure in the mRNA Since reverse transcription and PCR are performed in one step we recommend to use the reverse PCR primer as the gene specific primer for reverse transcription Recommended Control Reactions No Template Control NTC no template control reactions are recommended in each experiment to screen for contamina
8. tion of reagents or false amplification No RT Control no RT control reactions are recommended for each experimental sample by omitting reverse transcriptase from the reaction The no RT control should generate no signal if the primers are specific for the cDNA and does not amplify genomic DNA Use of the ROX Reference Dye ROX reference dye is included in this kit and may be added to compensate for non PCR related variations in fluorescence Addition of the reference dye is optional Optimizing the ROX dye concentration within the qPCR reaction is an important aspect of setup Too much ROX in the qPCR reaction will reduce background but also makes a low target signal difficult to distinguish from background Conversely too little ROX can increase background meaning that low or weak target signals can be lost For instruments that allow excitation at 584 nm such as Stratagene s Mx instrument and ABI 7500 firstly 1 10 dilute the ROX reference dye provided in the kit then begin optimization using 0 5 ul diluted ROX reference dye in 25 ul qRT PCR reaction For instruments that do not allow excitation near 584 nm such as ABI PRISM GENEAmp 5700 instruments begin optimization using 0 5 pl undiluted ROX reference dye in 25 ul qRT PCR reaction Reagent Preparation and Storage Thaw the tube containing 2x qRT PCR Reaction Mixture on ice and store it on ice while setting up the reactions 1 Ifthe ROX reference dye will be included in the react
9. y mix the reactions without creating bubbles since bubbles interfere with fluorescence detection Then centrifuge the reactions briefly BioChain Institute Inc Website www biochain com t 888 762 2568 f 510 783 5386 e mail info biochain com QCell Pro One Step qRT PCR SuperMix Kit 020107V2 3 Place the reactions in the instrument and run the appropriate RT PCR program Try the following protocol first and optimize the reaction conditions if needed PCR program for RT PCR ptf ee tS min OFF rr 95 C 10 min OFF This step inactivates the reverse transcriptase and activates the hotstart Taq DNA polymerase 10 minutes incubation is required to fully activate hotstart Taq DNA polymerase iae a Set an appropriate annealing tem perature for the primer set used 4 Dissociation Program for all PCR products Follow manufacturers guidelines for setting up dissociation depending on the instrument s software version Related Products QCell E va One Step qRT PCR SuperMix Kit Cat K5054200 K5054400 Eva QPCR SuperMix Cat K5052200 K5052400 Pro QP CR SuperMix Cat K5053200 K5053400 dNTP set for PCR Cat K6011100 PCR mix Cat 5051100 PCR Optimization Kit K5051100 Taq Polymerase Cat 7051200 RNA PCR ready cDNA and PCR ready genomic DNA References 1 Higuchi R Dollinger G Walsh P S and Griffith R 1992 Simultaneous amplification and detection of specific DNA sequences BioTechnology 10 413 417 2
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