I'm currently optimizing my primers at various annealing temperatures. Initially, I achieved bands at the target product size using a specific DNA sample. However, when I applied the same conditions to different samples, I observed no bands. I then re-ran the experiment on the original sample, which previously showed bands, but this time no bands appeared. I've repeated the experiment 2-3 times to rule out handling errors, but still no bands.
For reference, my reaction mix includes 12 µL of Master Mix, 4 µL of PCR water, 2 µL of DNA sample, and 1 µL of each primer, totaling 20 µL. Any suggestions on what might be causing these inconsistent results? I’ll share gel images showing some faint bands. But now I am not getting any bands.
hello Maira Zafar could you explain more detail about:
1. what you mean you optimized with specific DNA sample?
2. what different (real sample) you use? or what source is it (blood, soil, cell, etc)?
3. how you prepare/extract the sample DNA? And what about the concentration and purity A260/280 ratio?
My first concern is, your gel image which shown the expected target band was too weak and there was multiple band for optimization result. The smear result also could be caused by the quality of your DNA sample.
1. Specific means I was using one of my DNA samples to optimize my primers. My sample size is 70. It's from Breast Cancer Biopsies (FFPE blocks).
2. Different sample means, when I tried to use other 3 or 4 DNA samples from those 70 extracted DNA samples using one of the annealing temperature where I got band. It was 60° where band was somewhat visible than other temperatures where bands are faint.
3. I used 3 days DNA extraction protocol which is optimized by our seniors. And the samples I am using for optimization have concentration greater than 3000 ng/ul, and ratio around 1.6, 1.77 etc shown by nanpdrop. I diluted the DNA by DNA dilution factor formula using these and then took 2 ul from each sample.
I performed a gradient PCR, with the temperature varying for each PCR mix in each well. The most prominent band appeared at 60°C, so I set the annealing temperature at 60°C. However, when I used other DNA samples with this annealing temperature, I did not get any bands. This is with the wild-type primer.
Maira Zafar Thanks for additional information.
actually, I'm surprised with the DNA concentration you get from FFPE block sample, it's quite high concentration. Because normally FFPE treatment sample causing a lot of DNA degradation and crosslinking effect because of the formalin, so the usual concentration won't that much, in my experience usually 50-200 ng/ul. How about the A260/230 ratio?
I don't know the dilution you make and total DNA for that 2 ul, but try to input total DNA around 50-100ng per 50 uL PCR reaction, increasing PCR volume reaction can dilute the effect of inhibitor. Also, you can try using another PCR mastermix that tolerant to inhibitor. Because FFPE sample can cause a lot inhibitor that may differ from one sample to another.
Thank you very much for your response and helpful suggestion! I’ll give this a try and see how it works!
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