I am trying to design a primer for a specific gene and the Tm is coming near to 60 degrees when I select a minimum of 30-35 bp, for forward as well as for reverse primer. As the template is too long I think it might lead to non-specific binding, what are the possible solutions for this problem? I am using Snapgene to design primers for cloning
There is no reason why 30mers should not be very good primers. In increasing the annealing temperature you have made non specific priming less likely and I would expect the primers to work as expected. If your primers and template are very AT rich then you may need to minimise secondary structure in both by using DMSO additive in the pcr reaction
The chances of non-specificity increase with oligonucleotides of short lengths not with long ones. Primer-BLAST will give you many sets of primers with lengths of 18-30 nucleotides and Tm 60 degrees. The length of 30 nucleotides will work. There will be no problem.
Folks routinely use primers that are only 20 base pairs and can get specific amplification from genomic DNA. 30-35 bases will give you even greater specificity for your region of interest.
Standard Tm is 60*C, but it can range higher or lower if needed.
I'm not seeing any concerns with your design. What exactly do you think is the problem?
1. Adjust primer length: Increase or decrease primer length slightly.
2. Evaluate Tm prediction methods: Use different algorithms or tools for Tm calculation.
3. Adjust primer sequence: Make small modifications to fine-tune Tm.
4. Check for secondary structures: Identify and minimize hairpins or self-complementarity.
5. Consider primer placement: Avoid repetitive or variable regions.
6. Follow primer design guidelines: Adhere to established recommendations.
7. Validate and test primers experimentally.
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