| Protocolforhuman | Protocolformouse |
Table:Validatedprimersetsforhumanandmousehousekeepinggenesandtheirexpressionlevels.
*Summaryoftheexpressionlevelsofgenesincomparisonwith18SrRNAusingoursensitivityhumanormousereferenceCDNAandourproprietarySYBRgreenmastermix. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ebiomall.com
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原位杂交引物要求:
1. 为了避免检测mRNA时基因组序列的干扰,设计探针时尽量选择在跨内含子的部位。
2. 通常探针的长度(既PCR产物长度)以100-400bp为宜,过长不易穿透组织,杂交效率减低;过短特异性低。
而Realtime引物设计:
1.产物长度80-150bp为宜,可延伸到300bp。
而且你做实时定量的时候,还要做标准曲线,确定引物的扩增效率好不好。
引物设计略有不一样,但都可用Primer5设计。
It is designed to address the challenge of pathogen detection, species identification and taxa discrimination using TaqMan and molecular beacon assays. With ClustalW multiple sequence alignment at its core, AlleleID can help design universal or taxa/species specific primers and probes. All major qPCR assays including molecular beacons, TaqMan, FRET or SYBR Green are supported. It uses BLAST search to assure specificity. To assure assay success, AlleleID uses template folding to avoid locating primers in the secondary structures of the template.
With AlleleID, you can avoid gDNA by designing primers/probes across exon/exon boundaries. The intron exon regions are identified by interpreting GenBank annotations or by specifying them manually.
To get started quickly with AlleleID, please download and listen to the multi-media tutorial:
http://www.PremierBiosoft.com/DATAFILES/AL100Tour.exe.
Then please download the demo version of the program from:
http://www.premierbiosoft.com/DATAFILES/AlleleIDTour.exe
看引物能不能特异性扩增,你可以检查一下扩增后的熔解曲线,一般单一的峰是特异性引物,如果有双峰那就是扩出来了杂带
http://medgen.ugent.be/rtprimerdb/


