Recently we tried to standardize Real Time PCR from a clinical samples extracted cDNA or DNA and upon quantification results ends with A260/280=2.0, 2.5, 2.6 and the concentration maximum of 10ng/micro L.
In order to standardize, Before Proceeding this , i want to get the suggestion, whether this cDNA with higher protein/solvent content will affect the Real Time PCR ?
Thanks Tina,
But for the information, i have a quantified DNA only, not the cDNA, will the DNA with salt and other impurities will affect the Real Time PCR results ?
Thats my query?
Thanks in Advance.
yes it will effect the qPCr result so whaty i will suggest is if you can go for ethanol based percipiation purification, and remove maximum salt, but your reading of 2.0 is ok in its quality what yoiu dont have is the quantity, but for each pcr reaction you need only 10 to 50 ng of styarting concerntraion.
what kind of study you r performing
standrad or relative that also matters.
If you enough vol of your DNA then what i will suggest is even without purification go for 5 repliocates of the same sample and apiritate at least 15 times before alloting the sample to qPCR plate
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