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蚂蚁淘在线 / 品牌中心 / Alstembio / Decalstm&trade；支原体检测试剂盒/MD200/200 rxns

Decalstm&trade；支原体检测试剂盒/MD200/200 rxns

价格
￥6200.00
货号：MD200
浏览量：127
品牌：Alstembio
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商品描述
 

MycoDect™MycoplasmaDetectionKit:#MD050

 

ProductDescription

Mycoplasmasaresmall,roundorfilamentousprokaryoticorganismsthatlackcellwalls.Ithasbeenestimatedthatatleast15%-35%cellculturesarecontaminatedwithmycoplasma.Mycoplasmacontaminationcanmodifymanyaspectsofcellphysiologywithoutcausingobviousmediumcloudinessandapparenteffectoncellgrowth.Mycoplasmacontaminationaffectsupto80%ofcontinuouscellcultures.Ifundetected,mycoplasmacontaminationcanhavesignificanteffectsonthequalityandreliABIlityofyourcellculturepreparations.Duetotheirsmallsizeanddeformability,mycoplasmascanpassthrough0.22µmfilter.Thelackofcellwallmakesmycoplasmaunresponsivetocommonantibioticsthattargetcellwallsynthesis.Itisessentialthatallcellstocksandnewculturesenteringafacilityaretestedforthepresenceofmycoplasmaandallcellculturesshouldbetestedroutinely(e.g.onceevery2-3months)inordertomaintainamycoplasma-freeenvironment.
Manymethodsareavailablefordetectionofmycoplasma,includingisolationinbroth/agarculture,directorindirectfluorescencestaining,ELISA,immunostaining,directorindirectPCR.Amongthosemethods,directPCRisthemostsensitive,specificandconvenientmethodwhentheprimerdesignisoptimized.
TheMycoDect™MycoplasmaDetectionKitisdesignedtodetectmycoplasmainfectionincellculturesinlessthantwohours.Itcandetectmycoplasmafrombothcelllysatesandcellculturemedia.Thesensitivityisupto10-20copiesoftargetDNA,whichtranslatestolessthan10mycoplasmaspecies.Thiskitdetectsthetopfivecommonspeciesofmycoplasma,M.Hyorhinis,M.Arginini,M.Orale,M.Fermentans,A.laidlawii,whichrepresent98%oftissuecultureinfections.ItcanalsodetectMycoplasma,Acholeplasma,UreaplasmaandSpiroplasmafromothergenera.

KitComponents

  • MycoDect™PCRMix
  • MycoDect™PrimerMix
  • PositiveControl
  • NegativeControl
  • LysisBuffer

ExampleofExperimentalResults

Sample1showsseveremycoplasmacontamination.Sample2givespositiveresultsformycoplasmawithboth270and150bpbands.Sample3givesnegativeresultswith150bpbandonly.
 
ProductSpecifications
ProductNameMycoDect™MycoplasmaDetectionKit
Catalog#MD050
Size50reactions(MD050)/200reactions(MD200)
ShippingDryiceorblueice
StorageandStabilityStoreat-20°C.Thisproductisstablefor12monthswhenstoredasdirected.
QualityControlEachlotofMycoDect™MycoplasmaDetectionKitisfunctionallytested.
RestrictedUseForResearchUseOnly.Notforuseindiagnosticortherapeuticprocedures.
ProtocolDownload
 
  • ExperimentalProcedure
  • ExplanationofResults

CellsculturedintheabsenceofantibioticsformorethanthreedayscanenhancethePCRsignal.Mediasampleshouldbederivedfromcellsthatareatleast80%confluent.

SamplePreparationfromCellLysate
  1. Aspiratemediumfromattachedcells,andrinsecellstwicewithDPBS.
  2. Add0.2mLoftrypsinperwellof24-wellplate,andincubateat37°Cfor2-3minutes.
  3. Add0.5mLof10%FBS/DMEMmedium,andtransferthecellsUSPensiontoa1.5mLmicrocentrifugetube.
  4. Centrifugethis1.5mLtubecontainingthecellsuspensionat300xgfor5minutesatroomtemperature.
  5. Aspiratethesupernatant.Washthecellpelletwith1mLDPBS.
  6. Centrifugethetubeagainat300xgfor5minutesatroomtemperature.
  7. Aspiratethesupernatant,andresuspendthecellpelletin100μLlysisbuffer.
  8. Lyseatroomtemperaturefor10min.
  9. Heatthelysatesat95°Cfor5min.
  10. Centrifugethelysatesat15,000xgfor5minandcollectsupernatant.
  11. Take1-2μLsupernatantastemplateforPCRreaction.
SamplePreparationfromMedia
  1. Collect100µLofcellculturemediumtoamicrocentrifugetube.
  2. Centrifugeat15,000xgfor5min.
  3. Heatthecellculturemediumat95°Cfor5min.
  4. Take1-2µLmediumastemplateforPCRreaction.
PCR

PCRsetupwithTaqpolymeraseisshownbelow.Afinalvolumeof20μLisrecommendedforeachreaction.

 TestSample/μLPositiveControlReaction/μLNegativeControlReaction/μL
MycoDect™PrimerMix151515
MycoDect™PCRMix444
Sample1--
PositiveControl-1-
NegativeControl--1
InTotal202020

PCRProgram

StepTempTime
InitialDenaturation95°C4min
30Cycles95°C15s
56°C30s
68°C30s
FinalExtension68°C5min
Hold4°C 

PCRProductDetection

Foroptimalseparationbetweenthemycoplasmabandandthecontrol,werecommend2%agarosegelforelectrophoresis.

  1. MixthefinalproductsofeachPCRreactionwithgelelectrophoresisloADIngbuffer.
  2. Loadeachsampleintoindividualwellsoftheagarosegel,includingpositivecontrol,negativecontrol,andDNAladder.
  3. Electrophoreseattheconditionsrecommendedbythegelboxmanufacturer.
  4. Visualizethebandswithethidiumbromide.

Limitations

  • Useribonuclease-free(RNase-free)reagentsandsupplieswhenrunningthisassay.
  • Donotmixorsubstitutereagentswiththosefromothersourcesorlots.
  • Thekitshouldnotbeusedbeyondtheexpirationdateonthekitlabel.
  • Anyvariationindiluents,operator,pipettingtechnique,washingtechnique,incubationtimeortemperature,andkitagecanalterassayperformance.
  • Thisassaydetectsthefollowingspecies:M.hyorhinis,M.arginini,M.fermentans,M.orale,M.pirum,M.hominis,M.salivarium,andA.laidlawii.
  • Anegativeresultdoesnotindicatethatotherspeciesofmycoplasmaareabsent.Additionally,mycoplasmamaybepresentatlevelsbelowthedetectablelimitsofthiskit.
  • CellculturesthatarevisIBLycontaminated(i.e.,turbidityandyellowmedia)areprobablyduetoE.coliorfungalinfectionandshouldnotbetested.Visiblycontaminatedcellculturesshouldbediscardedandfreshculturesshouldbestartedfromfrozenstock.
  • Thisassaycannotbeusedformycoplasmaspeciesidentification.
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