cDNA - amonut to use (Sep/07/2007 )
I'm wondering how much cDNA to use as a templete for my RT reactions. I'm new to molecular biology and I'm wondering if it's common practice to use a spectrophotometer to determine the concentration of your cDNA sample and then adjust your volume to the amount you want. It's important I use the same amount of cDNA in each of my experiments.
Any help you can lend to a FOB would be greatly appreciated.
I spec my RNA and use the same amount in each RT reaction. From there I think you can assume the efficiency of RT will be the same across all samples... besides you will likely normalize with a house keeping gene as well.
I usually use 400ng of cDNA per reaction in all my PCRs. As Brandi, I measure a sample of my RNA before a do RT and assume the amount of cDNA will be the same. Depending on what you will use your cDNA (I use it for relative quantification in real time PCR) you will control your cDNA by using an endogenous gene which should have the same CT in all assays.
i use 100 pg cDNA sample as template in my real time pcr