I recently designed the qrtpcr primers from the 3'utr of the plant's genomic DNA keeping in mind that the annealing temperature should be 60 degree. Once I received the primers first to optimise their annealing temperature I set a gradient PCR on the genomic DNA itself (the concentration used was 25ng/ul) from temperatures ranging from 53-60 and 49-59 but I didn't see any band in the earlier temperature range but I did see some specific+ non specific band atleast for one amplicon in the later temperature range. Now how is this possible if the temperature that I chose while designing the primers using different softwares is sooo far fetch from the temperature where I am seeing the band. And why is it so? Still I am not sure whether the band that I feel is specific or non specific?? Please suggest me what do I need to change.
Note: I already did BLAST as well while designing the primers
Whats the sizes of the unexpected bands? If something small, can the non-specific bands be primer dimers / heterodimers? Use these softwares this screen for homo/heterodimers:
https://www.thermofisher.com/tr/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/multiple-primer-analyzer.html
&
https://www.idtdna.com/pages/Support/FAQs/how-do-i-use-the-oligoanalyzer-tool-to-analyze-possible-hairpins-and-dimers-formed-by-my-oligo
The annealing temperature for PCR primers is a critical parameter that directly influences the specificity and efficiency of the PCR reaction. The annealing temperature should ideally be set a few degrees below the melting temperature (Tm) of the primers to promote specific binding to the target DNA sequence.
The Tm of a primer is influenced by factors such as its length, nucleotide composition, and salt concentration in the PCR reaction. Several online tools and software programs are available to calculate the Tm of primers based on these factors.
As a general guideline, the annealing temperature for PCR primers is often set between 3 to 5 degrees Celsius below the Tm of the primers. However, it may require optimization depending on the specific primers, target sequence, and PCR conditions.
During PCR optimization, a gradient PCR or temperature gradient PCR can be performed to determine the optimal annealing temperature. This involves testing the PCR reaction at a range of annealing temperatures to identify the temperature that yields the highest amplification efficiency and specificity.
It's important to note that while lower annealing temperatures can increase nonspecific amplification, higher annealing temperatures may reduce PCR yield. Therefore, finding the balance between specificity and efficiency is crucial for successful PCR amplification.
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