User`s Manual
Contents
1. cell lysate from 64 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 K5054200 Reagents are sufficient for 200 assays Meni 2 y l PartNo 1 Cell Lysis Buffer K5054200 1 2 Eva qRT PCR Reaction Mixture 2x containing 1 25 ml x2 K5054200 2 Eva Dye and Hotstart Taq DNA polymerase K50542003 6 Nuclease Free PCR Grade Water Catalog Number K5054400 Reagents are sufficient for 400 assays bitem aeee e a a a PartNo 1 Cell Lysis Buffer K5054400 1 2 Eva qRT PCR Reaction Mixture 2x containing 1 25 ml x4 K5054400 2 Eva Dye and Hotstart Taq DNA Polymerase BioChain Institute Inc 3517 Breakwater Avenue Hayward CA 94545 Tel 888 762 2568 Fax 510 783 5386 E mail cs biochain com Website ww biochain com 3 QCell Eva 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 Eva Dye and the ROX reference dye are light sensitive and should be kept away from light whenever possible BioChain Institute Inc 3517 Breakwater Avenue Hayward CA 9452. 45 Tel 888 762 2568 Fax 510 783 5386 E mail cs biochain com Website ww biochain com 4 QCell Eva One Step qRT PCR SuperMix Kit 020107V2 Protocol Primer Design Design QPCR primers to generate amplicons of lt 250 bp Since the cell lysate contains genomic DNA the primers should be designed to amplify cDNA but minimize amplification of genomic DNA It is useful to choose primers that span an exon exon boundary in the target mRNA or choose primers that flank a large intron If possible design primers 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 contamination 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
3. QCell Eva One Step qRT PCR SuperMix Kit 020107V2 User s Manual and Instructions Product QCell Eva One Step qRT PCR SuperMix Kit Catalog Number K5054200 K5054400 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 Eva fluorescent dye and hotstart Taq DNA polymerase in a separate 2x reaction mix optimized for qRT PCR Both cDNA synthesis and PCR are performed in a 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 qRT PCR bypassing RNA isolation procedure The passive reference dye ROX is included in a separate tube to make the QCell Eva One Step qRT PCR SuperMix adaptable for many real time QPCR platforms Human GAPDH primer set for RT PCR is also included in the kit as a control This primer was designed to span an exon exon boundary in the human GAPDH cDNA which can eliminate the undesired amplification of genomic DNA in the RNA or cell lysate BioChain s QRT PCR SuperMix contains BioChain s Taq polymera
4. 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 Avoid direct light in preparation of the PCR reaction mixture because Eva Dye is light sensitive 1 Ifthe ROX reference dye will be included in the reaction 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 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
5. aq DNA polymerase 10 minutes incubation is required to fully activate hotstart Taq DNA polymerase 40 50 60 C a Set an appropriate annealing temperature for the primer set used 4 Dissociation Program for all PCR products BioChain Institute Inc 3517 Breakwater Avenue Hayward CA 94545 Tel 888 762 2568 Fax 510 783 5386 E mail cs biochain com Website ww biochain com 6 QCell Eva One Step qRT PCR SuperMix Kit 020107V2 Follow manufacturer s guidelines for setting up dissociation depending on the instrument s software version QRT PCR Setup and Cycling Program for human GAPDH control primer set amplicon size 226 bp 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 Reverse Transcriptase RNase Inhibitor Mixture Human GAP DH primer set 25x Reference Dye ROX Template Nuclease free PCR grade water See page 5 Use of the ROX Reference Dye 2 PCR program for amplification of human GAP DH amplicon 3 Dissociation Program Follow manufacturer s guidelines for setting up dissociation depending on the instrument s software version Related Products QCell Pro One Step qRT PCR SuperMix Kit Cat K5055200 K5055400 Eva QPCR SuperMix Cat K5052200 K5052400 Pro QPCR Supe
6. ce 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 qRT 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 feral KL Inhibitor Mixture 150 200nM PCR forward primer 150 200 nM ROX Reference Dye 0 5 ul pO Template cell lysate or RNA 1 25 y O Nuclease free PCR grade water Addupto25ul O OS See page 5 Use of the ROX Reference Dye P 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 250 ng PCR reverse primer 150 200 nM V2 2 Gently mix the reactions without creating bubbles since bubbles interfere with fluorescence detection Then centrifuge the reactions briefly 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 aC AS min ORE 1 95 C 10 min OFF This step inactivates the reverse transcriptase and activates the hotstart T
7. ell K562 cells were lysed according to the cell lysis protocol 4 fold serial dilution of cell Isyate were prepared from 64 cells to 1 cell GAPDH gene expression was detected using BioChain s QCell Eva qRT PCR kit on Stratagene s Mx3005P instrument Efficiency as measured from standard curve was 102 3 with a R value of 1 000 Features J RNA in one step format and liquid handling steps Applications Real Time RT PCR Gene expression profiling Gene knockdown verification Array Validation Flexible and convenient quantitating gene expression in cells without isolating RNA or Save time quick cell lysis procedure and ready to use supermix reducing setup time High Sensitivity qRT PCR from as low as 1 cell or 1 pg total RNA Versatile compatible with a wide variety of cell lines BioChain Institute Inc 3517 Breakwater Avenue Hayward CA 94545 Tel 888 762 2568 Fax 510 783 5386 E mail cs biochain com Website ww biochain com 2 QCell Eva One Step qRT PCR SuperMix Kit 020107V2 Description Components in this kit are prepared with pure chemicals according to our proprietary technology QCell Eva 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 intercalator format Quality Control 1 kit of this lot has been tested for quantitating human GAPDH gene expression in a serial dilution of
8. 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 500 This is a general guideline For some cells lines 500 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 BioChain Institute Inc 3517 Breakwater Avenue Hayward CA 94545 Tel 888 762 2568 Fax 510 783 5386 E mail cs biochain com Website ww biochain com 5 QCell Eva One Step qRT PCR SuperMix Kit 020107V2 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 i
9. rMix 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 Biotium Inc at http www biotium com product product_info allcolor paf 2 Higuchi R Dollinger G Walsh P S and Griffith R 1992 Simultaneous amplification and detection of specific DNA sequences BioTechnology 10 413 417 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 BioChain Institute Inc 3517 Breakwater Avenue Hayward CA 94545 Tel 888 762 2568 Fax 510 783 5386 E mail cs biochain com Website ww biochain com 7
10. se 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 And the real time RT PCR buffer is specially formulated to provide superior specificity and increase reverse transcription and amplification efficiency Eva Dye Eva Dye binds double stranded DNA Detection is monitored by measuring the increase in fluorescence intensity throughout the cycle Eva Dye has higher affinity to double stranded DNA than SYBR Green dye and shows stronger fluorescence intensity than SYBR Green upon binding to DNA Eva Dye is more stable than SYBR Green and the absorption and emission spectra of Eva Dye are very similar to SYBR Green Dye or FAM so the same optical setting for SYBR Green Dye or FAM can also be used for Eva Dye BioChain Institute Inc 3517 Breakwater Avenue Hayward CA 94545 Tel 888 762 2568 Fax 510 783 5386 E mail cs biochain com Website ww biochain com 1 QCell Eva One Step qRT PCR SuperMix Kit Fluorescence dRn tt pp aee a a a a y 020107V2 aooo 2 4 6 8 10 12 14 16 18 20 Cycles 22 24 26 Y 3 269Log x 31 11 R 1 000 Efficiency 102 3 Ct Rn 10 Initial Quantity Cell number Figure 1 BioChain s QCell Eva One Step QRT PCR SuperMix provides sensitive detection down to a single c
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