Hi,

I would suggest that there is something wrong with your cDNA stock (sample 1) or your GAPDH assay. If this stock truly contains 70ng/µl cDNA I would expect a Ct value in the range of 20-25 for GAPDH. Is the melting curve and the PCR product in the gel okay? Have you any positive control? Maybe a cloned GAPDH sequence to check your assay?

Best,

Norman

Shawyun Khosh how did you prepare your standard curve?

Hi Norman Hafner , I meant to say that my stock cDNA concentration reading was 771.5 ng/ul. I performed a melting curve analysis on the program and it looked like it was ok.

The only control I had was my NoRT control.

I think I may have made an error when creating my cDNA dilutions.

Giorgia Pertile I did a 10 fold dilution series using a mixture of 6 different cDNA samples I had. My stock concentration is 771.5 ng/ul. I had 4 different dilutions and then 4 replicates of those dilutions.

Shawyun Khosh how much replicate of your standard curve did you prepare?

When you prepared your standard, you centrifuge and mix well the sample before each serial dilution?

Hi, how did you quantify your cDNA? Had you purified it after synthesis?

Giorgia Pertile Norman Hafner It looks like it may have been due to a simple pipetting error. Because I did not properly aspirate my mixtures, I had varying responses. I ran another reaction today with my concentrated cDNA stock along with my NoRT and I got consistent results! I have attached an image here.

I did include a primer with my NoRT nd NT because I wanted to see if my pipetting was precise.

Good news