OptimizePCRreactionsthatusedifferentenzymesthantheFailSafe™System(e.g.Taq,Pfu,andTth).
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Forrapid,dependableoptimizationofPCR,weencourageyoutotrytheFailSafe™PCRSystemtoobtainthebestPCRresultswithanytemplateupto~20kb.However,ifyouprefertouseanotherenzyme,EpicentrealsohastwoMasterAmp™PCROptimizationKitsthatwillhelpyoutoobtainthebestpossIBLePCRresultswiththermostableenzymesfrequentlyusedinPCR,suchasTaq,Pfu,Tth,TflDNApolymerases.TheseMasterAmpPCROptimizationKitscontainaseriesof12MasterAmpPCR2XPreMixesthathavebeenmeticulouslytestedandrepresentacompleterangeofPCRconditionsfrequentlyencountered.EachMasterAmp2XPreMixcontainsallcomponentsneededforPCRexceptfortheprimers,thetemplate,andthePCRenzyme. First,usetheMasterAmp™PCROptimizationKit(Cat.No.MO7201)toperformPCRwithyourtemplate,primers,andeachofthe12MasterAmpPreMixes.SelectthePreMixthatgivesoptimalresults.Then,usethesameMasterAmpPCRPreMixtoobtainreliable,consistentPCRusngthesameDNAtarget/primerset. | STEP1.FindtheoptimalMasterAmpPCRPreMix.DoPCRwithyourtemplate,primersandPCRenzymeinthe12PreMixes. | |
A.A75%-GC-richApoEgeneregionwasamplifiedintotalhumangenomicDNAusingPreMixesA-L.MasterAmpPCRPreMixKproducedoptimalresultsinStep1. | ||
STEP2.UsethesameMasterAmpPCRPreMixtoobtainreliable,consistentPCRofthesamesequence. | ||
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组织用Trizol法提取总RNA浓度、纯度都很好,但逆转录后跑PCR,CT值偏高,内参的在22-27之间,目的基因在30-36之间,不知道是怎么回事,如何来解决,求高手赐教。我用的Takara的试剂盒。
这样设想的前提是因为,普通Taq酶也具有5‘-3’外切酶活性,如果可以的话,与普通PCR相比,反应体系需要做什么样的改动呢?
用的是ABI的机器。
如果要测量细胞表面2个受体的比率,最好用流式细胞仪来测量
标准品的荧光强度和已知的浓度作图,可以得到一条标准曲线。而待测样本的荧光强度测出以后,就可以在标准曲线上算出样本浓度了。
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?
C代表的是cycle,循环数目,T代表的是threshold,阈值。
所以表达量越少的话,需要越多的循环才能扩增出来。也就是CT值越高
有大神做过人外周血全血miRNA的提取及后续PCR实验的吗?目前课题实验遇到瓶颈,求助大神!!!是否miRNA的PCR验证在全血做不出?必须要用血浆或者血清?目前血浆、血清也有部分样本。。。。有大神指条明路吗?
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