We performed qPCR analysis using SybrGreen dye, where the template was gDNA, on the Applied Biosystems 7500 Fast Real-Time PCR System (software ver 2.3). The reaction conditions were optimized using standard PCR conditions (denaturation at 95°C for 60s, annealing at 58°C for 30s, elongation at 72°C for 30s). First, these conditions were tested. We also tested the default program for SybrGreen (2 step, 60°C annealing + melting curve, photo1,2) and many other combinations each time, without obtaining any curves (photo3). I know that the reaction itself, reagents and temperature profile is well optimized because after the reaction, we made an electrophoresis of the product (photo4).
Do anyone have experience with this software? Is this software bug (do you press any extra option?) or do I make something wrong? Additionally, is it possible to preview the amplification plot such that it is presented on a linear and not a logarithmic scale during reaction? I will be grateful for any tips.
The agarose gel shows you do have amplification, so that rules out issues with the PCR program.
SYBR reagents are very light-sensitive and will degrade from repeated freeze-thaws and from exposure to light. Get a new tube and try again.
Also, double-check that you are asking the machine to measure the wells that actually have your sample. It's a silly thing, but I've accidentally put the tubes in the first column (A1, B1, C1, etc.) instead of the first row (A1, A2, A3, etc.)
Good luck!
When performing a qPCR run, I always choose the delta-delta-CT option from your second picture. I think that's why you have the final product but the real-time curve.
This could happen if the machine is reading the ROX value from your ctr.
You need to go to "set up" then "assign targets to wells", for the endogenous ctr. select "none" in the reference. Then click on "analyze". Your amplification curves should appear.
If again no amplification, then you need to increase the amount of your starting cDNA samples or increase the number of cycles to 45
+
choose the delta-delta-CT option from your second picture as mentioned by Yi-Chun Cheng
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