Is it essential to eliminate all the 5 parameters of Gene Runner : hairpin loops, dimers, bulge loops, internal loops and match sites of primers for mutant generation?
I'm manually designing primers and checking it with Gene Runner, but can't able to eliminate all these possibilities.
Thanks in advance for suggestions.
Eliminating all potential issues such as hairpin loops, dimers, bulge loops, internal loops, and match sites of primers is indeed important to ensure the effectiveness and specificity of your primers. However, it's not always possible to completely eliminate these factors, especially in manual primer design.
Here are some suggestions to optimize your primer design process:
While it may not always be possible to eliminate all potential issues completely, careful optimization and validation can greatly improve the performance of your primers for mutant generation.
Why are you designing by hand? Use a primer designing tool like the one in Primer BLAST.
Generally, in PCR, I have broken the rules before and used the primers which the softwares did not recommend. It mostly worked fine.
(I only had problems in a specific situation when I used certain modified bases, which is probably not your case).
But I am not sure how your mutant generation works, if there are other requirements.
Eliminating the possible errors, will ensure the specificity of the primers
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