I have tried aII combinations of primers and Temp. for genomic DNA standarts, but I havent got a good efficency, even tho the primers have been reported having a very good efficency using genomic DNA. I have the information that my DNA couId be fragmented, but Im wondering that maybe this time I wiII extract RNA instead of DNA , to do a standarization with cDNA. Any recomendations or thoughts about it? which one of the tempIates have gave you better resuIts?
If the gDNA is fragmented, you could isolate new one and test your PCR again.
Also, there are a number of possible causes of poor PCR efficiency:
- Your samples may contain PCR inhibitors
- Your PCR primer and/or probe design may not be optimal
- Inaccurate sample and reagent pipetting
- The standard curve may not have been properly analyzed
http://www.lifetechnologies.com/de/de/home/life-science/pcr/real-time-pcr/qpcr-education/real-time-pcr-troubleshooting-tool/gene-expression-quantitation-troubleshooting/poor-pcr-efficiency.html
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