- OutstandingreliABIlityandreproducibilityinColonyPCR
- IdealforPCRgenotyping
- Convenient1XMasterMix:justaddacolony,primers,andcycle.
- HighThermostabilityandoptimizedformulationfor98Cdenaturationtohandlethetoughesttargets.
- ContainsGreentrackingdyefordirectgelloADIng
FreeSampleAvailable
Finally,aPCRenzymespecificallyformulatedforColonyPCR.
Reliableperformancefromcolonies.Bypassallcelllysisandsampleprepwithoutworryingaboutfailedreactions.Outstandingreproducibilitytogetyourampliconthefirsttime.(Figure1).
Extremethermostability. Cycleat98°Ctolysecellsanddenaturethetoughesttemplates.TheaddedPCRenhancerimprovesamplificationandthermostabilitytogiveyouthebestPCRproductpossIBLe.
Toughtemplatesarenomatch: GC-richDNAcanmakecolonyPCRthatmuchmore difficult–andCloneIDcanhandleitwithease.Cleanandrobustamplificationontargetswhereotherenzymesfallshort(Figure2)
Theeasiestworkflow. CloneIDisnotonlya1Xmastermixthatonlyrequiresacolonyswipeandprimers,butitalsocontainsaGreentrackingdyesoyoucandirectlyloadyourPCRproductontoagel.
Figure1.CloneID™1XColonyPCRMasterMixwasusedon96uniquecoloniesfromapetridishcontainingalibrarysample.Successratewas97%(93outof96)foramplification. |
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Figure2.CloneID™1XColonyPCRMasterMixiscomparedwithotherPCRproducts(CloneID™,Platinum®PCRSupermix,OneTaq®,KAPA2G)claimingstrongColonyPCRperformance.The12targetsaregenesfromCellulomonasfimi,aspecieswhosegenomeis75%GC,whichwereclonedintoastandardplasmid. |
ORDERINFORMATION
CloneID™1XColonyPCRMasterMixisprovidedina1XMasterMixformatthatwithstands98°Cdenaturation.Allrequiredbuffercomponents,enzyme,Mg++anddNTP’sareallpresentinoptimizedconcentrations.TheproductalsocontainsGreentrackingdyeforconvenientgelloading.Onlyasmallamountofwholecellsfromacolonyandprimersarerequiredtosetupthereaction.Pleaseseetheusermanualforcompositioninformation.Standardreactionsizeis25µL.
ThisproductisprimarilyintendedforE.colicoloniesorpurifiedDNAtemplates.CloneIDhasnotbeentestedfordiverseorganisms.
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荧光定量PCR数据如下表,想通过GraphPadPrism5软件构建柱状图,但不知道是将2-△△Ct数据导入GraphPadPrism5软件,还是将Mean和SD导入GraphPadPrism5软件中。请大神赐教。另外Mean和SD是求的△△Ct的还是2-△△Ct的?
PCR(聚合酶链式反应)是利用DNA在体外摄氏95°高温时变性会变成单链,低温(经常是60°C左右)时引物与单链按碱基互补配对的原则结合,再调温度至DNA聚合酶最适反应温度(72°C左右),DNA聚合酶沿着磷酸到五碳糖(5'-3')的方向合成互补链。
恒温PCR和实时荧光定量PCR的不同,是不同在实时荧光定量PCR的系统中加入了荧光染料(SYBR Green 或Taqman 探针等等)。以SYBR Green为例,这种染料可以结合在双链的DNA上,当PCR不断进行时,每一次退火生成的双链DNA也在增加,荧光染料结合也越多,荧光也越强。在机器中有一个探测荧光的探头,可以定量检测荧光的强度,转换成数值。这样就可以实时记录反映体系中DNA的反应情况。
荧光PCR更有优势,因为荧光PCR灵敏度高于恒温PCR,同样价格也高一些。
两步法,是因为设计引物的时候把退火温度设为酶的工作温度,而且定量PCR的产物都很短,需要的时候都短,所以两步法更方便。三步法的话,花的时间长,不利于快速实验。
另外还有几个问题。就是跑出来的结果RFU居然有负值,这是怎么回事呢?几乎在0附近,但是在溶解曲线求导前的那个曲线。RFU又是从2400+开始下降的,这是为什么呢?
暂无品牌问答