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template concentration qPCR


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#1 carry

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Posted 08 May 2009 - 07:08 AM

Hi,
I have a question regarding the concentration of template per reaction in qPCR. I usually use 250ng of mRNA to make cDNA and then use 1.2ng of template(normalized from mRNA concentration). I would like to know the minimum template concentration per reaction and also if using more mRNA has any effect on cDNA synthesis efficiency. I'd certainly appreciate if some could point me to nice working protocol for the complete process (mRNA to cDNA to qPCR).

Thank you in advance.

#2 asdfyrr

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Posted 08 May 2009 - 08:24 AM

I use the "superscript III First strand synthesis " kit from invitrogen to synthesis cDNA from mRNA with random oligo(dT) primers and around 500ng of mRNA.

afterwards i dilute the resulting cDNA 1:3 and the use this concentration as the starting point of a dilution row that i then analyse qith qPCR.

works fine for me




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