A quick question about series dilution - (Sep/08/2008 )

Hi all,
I confused about the series dilution of cDNA. I was told to do a 10-fold dilution of cDNA and then do real time PCR using these cDNA. Then make the graph to show the slope/efficency is within 95%-105%. But is this for the quanlity of primers or cDNA template? Do I have to do this series dilution experiment every time when I use a new cDNA template or new primers? This will use a lot of reagents.


Thank you.

Hi Yang,

Since you are only diluting the cDNA, the slope of your graph will also be for cDNA. The amount of primer added should be the same for all dilutions. Whether you will have to repeat it every time you get new cDNA or primers, really depends on your supervisor and the experiment you are running. Usually for such a standard curve, you will need to run triplicates with 3-5 dilutions, range about 50 pg to 500 ng. RTPCR kits are not that expensive. Its mostly just laborious. I would use a premix and then add primers, aliquot, and then add the dilutions individually. Have fun!

--SR