This is my first time doing at quantitative real-time PCR and I'm trying to do the absolute method where we plot the standard and then get the quantification using Unknown Ct value. My unknown gives Ct values but the standard does not. The amplification plots look weird. In a nutshell, I amplified the switch alpha region in WT CH12F3 DNA using Standard PCR and diluted it to make 10ng/ul to 100 ng/ul concentration gradient. Then I added Power-up SYBR green and volume was adjusted to 25 ul using water. I used triplicates for each concentration. For the unknown samples same amount of SYBR green was added and volume was adjusted to 25 ul. After qPCR i ran the products in a 1% agarose gel. I could see the amplified product in all wells. What is the reason for this weird plots for standard?
Although the signal level indicates low level of noise, if you see a product in gel, I think this might be some other issue. But as suggested by others melting curve could be informative.
We usually purify our PCR product for setting up standard series. Did you purify the PCR? How did you get the concentrations for the standard? Is it just based in the absorbance? with dNTP etc in the PCR absorbance does not give correct estimation.
If you didn't, purify the standard PCR you did to make the standard series first. Then measure absorbance of the PCR product. Based on the PCR product size, and MW of nucleotides, calculate the copy number according to Beer-Lambert and set the standard series based on copy numbers. You can try 10 - 100000 or one step higher. Good luck.
Nimanthi Jayathilaka Madam, I was able to solve this issue by reducing the standard amount to 0.1 ng to 1 ng. I was able to get a nice standard curve. To answer your question, yes I did purify the PCR product and the concentration was measured by Qubit. As you mentioned I calculated the copy number and found that my pull-down DNA has more copies of switch miu than the control, which was what I expected.
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