Techne ® qPCR test CaMV 35S promoter

1 CaMV 35S promoterTechne qpcr test 150 testsFor general laboratory and research use only1 Quantification of camv 35s promoter genomes. Advanced kit handbook to CaMV 35S promoter2 Quantification of camv 35s promoter genomes. Advanced kit handbook (CaMVp35S) profile possible whilst remaining specific to the CaMV p35S p35S sequences based on a comprehensive bioinformatics analysis. team will answer your of camv 35s promoter genomes. Advanced kit handbook Contents CaMV p35S specific primer/probe mix (150 reactions BROWN) FAM labelled CaMV p35S positive control template (for Standard curve RED) Internal extraction control primer/probe mix (150 reactions BROWN)VIC labelled as standard Internal extraction control DNA (150 reactions BLUE) Endogenous control primer/probe mix (150 reactions BROWN) FAM labelled RNAse/DNAse free water (WHITE)for resuspension of primer/probe mixes and internal extraction control DNA Template preparation buffer (YELLOW)

2 For resuspension of positive control template and standard curve preparationReagents and equipment to be supplied by the userReal-Time PCR InstrumentDNA extraction kitThiskitdesignedtoworkwellwithallproce ssesthatyieldhighqualityDNAwithminimalPC R 2x qpcr MastermixThis kit is designed to work well with all commercially available and TipsVortex and centrifugeThin walled ml PCR reaction tubes4 Quantification of camv 35s promoter genomes. Advanced kit handbook storage and stabilityThiskitisstableatroomtemperatur ebutshouldbestoredat-20 , from the positive sample ,concentration,andDNAintegrity(Aninterna lPCRcontrolissuppliedtotestfornonspecifi cPCRinhibitors). ,replacethetemplateDNAsamplewith RNAse/DNAse free range of testUnderoptimalPCRconditionsTechneCaMVp 35 Sdetectionkitshaveveryhighprimingefficie ncies of >95% and can detect less than 100 copies of target and disclaimersThisproductisdeveloped, ,1145 AtlanticAvenue,Alameda,CA94501orAppliedB iosystemsbusinessgroupoftheAppleraCorpor ation,850 LincolnCentreDrive,FosterCity, ,the5' ,210,015and5,487,972,ownedbyRocheMolecul arSystems,Inc, ,538,848,ownedbyThePerkin-Elmer is a trademark of Bibby Scientific ,683,195,and4,683, ,ABIPRISM GeneAmp andMicroAmp areregisteredtrademarksoftheAppleraGenom ics(AppliedBiosystemsCorporation).

3 BIOMEK isaregisteredtrademarkofBeckmanInstrumen ts,Inc.;iCycler isaregisteredtrademarkofBio-RadLaborator ies, ,TaqMan andAmpliTaqGold areregisteredtrademarksofRocheMolecularS ystems,Inc.,ThepurchaseoftheTechne PrimeProreagentscannotbeconstruedasanaut horizationorimplicitlicensetopracticePCR underanypatentsheld by Hoffmann-LaRoche of camv 35s promoter genomes. Advanced kit handbook of the testReal-time PCRACaMVp35 Sspecificprimerandprobemixisprovidedandt hiscanbedetectedthroughthe FAM , `-dyeanda3` , be detected on a range of real-time PCR controlForcopynumberdeterminationandasap ositivecontrolforthePCRsetup, , of camv 35s promoter genomes. Advanced kit handbook DNA extraction controlWhenperformingDNAextraction, are not present at a high + controlToconfirmextractionofavalidbiolog icaltemplate, biological prevention using UNG (optional)CarryovercontaminationbetweenP CRreactionscanbepreventedbyincludingurac il-N-glycosylase(UNG) (dUTP) during the Taq polymerase activation of camv 35s promoter genomes.

4 Advanced kit handbook - resuspend in waterVolumeCaMV p35S primer/probe mix (BROWN)165 lInternal extraction control DNA (BLUE)600 lInternal extraction control primer/probe mix (BROWN)Endogenous control primer/probe mix (BROWN)Pre-PCR packPre-PCR heat-sealed foil165 l165 lReconstitution ProtocolTominimizetheriskofcontamination withforeignDNA, cabinet. Filter tips are recommended for all pipetting each tube in a centrifuge before upon opening the ,according to the table below:To ensure complete resuspension, vortex each tube thoroughly.* ,away from the other extractionTheinternalextractioncontrolDN AcanbeaddedeithertotheDNAlysis/extractio nbufferor to the DNA sample once it has been resuspended in lysis NOT add the internal extraction control DNA directly to the unprocessed biologicalsample as this will lead to degradation and a loss in 4 l of the Internal extraction control DNA (BLUE) to each sample in DNAlysis/extraction buffer per DNA extraction according to the manufacturers the positive control template in the template preparationbuffersupplied, according to the table below.

5 To ensure complete resuspension, vortex the tube lPost-PCR heat-sealed foilComponent - resuspend in template preparation bufferVolumePositive Control Template (RED) *8 Quantification of camv 35s promoter genomes. Advanced kit handbook qpcr MasterMix1 lCaMV p35S primer/probe mix (BROWN)Final Volume1 l15 l10 lInternal extraction control primer/probe mix (BROWN)RNAse/DNAse free water (WHITE)3 lComponentVolume2x qpcr MasterMix1 lEndogenous control primer/probe mix (BROWN)Final Volume15 l10 lRNAse/DNAse free water (WHITE)4 lReal-time PCR detection each DNA sample prepare a reaction mix according to the table below:Include sufficient reactions for positive and negative For each DNA sample prepare an endogenous control reaction according to thetable below (Optional).

6 This control reaction will provide crucial information regarding the quality of thebiological lofeachmixintoindividualwellsaccordingto yourreal-timePCRexperimental plate set sample DNA templates for each of your lofDNAtemplateintoeachwell,accordingtoyo urexperimentalplateset each well is 20 to the table below:ComponentVolume2x qpcr MasterMix1 lCaMV p35S primer/probe mix (BROWN)Final Volume15 l10 lRNAse/DNAse free water (WHITE)4 l9 Quantification of camv 35s promoter genomes. Advanced kit handbook CurveCopy NumberTube 1 Positive control (RED)2 x 105 per lTube 2 Tube 3 Tube 4 Tube 5 Tube 62 x 104 per l2 x 103 per l2 x 102 per l20 per l2 per lStepUNG treatment (if required) **Enzyme activationDenaturationDATA COLLECTION *TimeTemp15 mins2 mins10s60s37 oC95 oC95 oC60 oC50 of standard curve dilution ) Pipette 90 l of template preparation buffer into 5 tubes and label 2-62) Pipette 10 l of Positive Control Template (RED) into tube 23) Vortex thoroughly4) Change pipette tip and pipette 10 l from tube 2 into tube 35)

7 Vortex thoroughlyRepeat steps 4 and 5 to complete the dilution lofstandardtemplateintoeachwellforthesta ndardcurveaccordingto your experimental plate set final volume in each well is 20 ProtocolAmplification conditions using Lyophilsed 2x qpcr MasterMix.* Fluorogenic data for the control DNA should be collected during this step through the FAM and VIC channels** Required if your Mastermix includes UNG to prevent PCR carryover contamination10 Quantification of camv 35s promoter genomes. Advanced kit handbook of ResultsInternal PCR controlTheCTvalueobtainedwiththeinternal controlwillvarysignificantlydependingont heextractionefficiency, , as a positive experimental material is present in the +ivePositivecontrolNegativecontrolIntern alcontrolTargetInterpretation+ive+ive+iv e+ive+ive+ive+ive+ive+ive+ive+ive+ive-iv e-ive-ive-ive-ive-ive-ive-iveExperiment failExperiment fail+ive-ive+ive-ive+ive-ive or +ive-ive or +ive-ive or +ive+ive-ive** (NCCT value sampleCTvalue).

8 WherethetestsampleispositiveandtheNCisde tectedmuchlater(deltaCT 5) (deltaCT<5)thenthe positive test result is invalidated and a negative call is the correct of camv 35s promoter genomes. Advanced kit handbook