I have been trying to use PCR to amplify cDNA using standard Taq polymerase. I am able to amplify beta actin, but not my target. Unfortunately, the Tm for my two primers is somewhat far apart (one is 57 and the other is 62). Based on the NEB calculator, I chose to start with annealing temperature of 52. After this failed, I tried to use the same annealing temp with more cycles, increased amount of template (1-4ul from a 20ul RT reaction), and increased primer concentration (200nM-600nM). Neither of these steps fixed the problem. Finally, I performed an annealing gradient from 50-60 degrees ... when the products were run on gel, it turned out ONLY a 55 degree annealing temp gave a band, which was not particularly strong. I have seen gels from annealing gradients that where the product appears to increase and then peaks at the optimal temp - is it also possible for the reaction to ONLY work at a specific annealing temp. Also, will increasing annealing time help at all? I am a novice at PCR so any advice will help!
Dear Patrick
You can read these papers :
https://www.drugdiscoveryonline.com/doc/using-gradient-pcr-to-determine-the-optimum-a-0002
http://www.techne.com/adminimages/A01_001B_PCR_Optimisation_gradient(2).pdf
https://primerdigital.com/pcr.html
So this paper was what actually brought about my question ... because when they do an annealing temp gradient, they see product at numerous temps, but more product at the optimal temp. Whereas I see product only at a specific temp, which makes me worried. I will look over this article more closely, thank you!
Dear Patrick, I think you use cDNA as a template. Beta actin is a housekkeping gene and amplification is normal. However, if your target gene is low expressed, you can or cannot see bands. Normally Real-Time PCR using SYBRGreen or prob based system are more sensitive to detect expression of genes.
Best regards.
Article Quantitative Analysis of Copy Number Variants Based on Real-...
Thank you Mert - so what your saying is that if I dont see clear band on agarose gel, perhaps I need to use RT-PCR to detect my product.
Dear Patrick, if it is possible, you perform Real-Time PCR for expression analysis.
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