it is my first time to use this technique (tetra-primer arms pcr). i want to know every thing about it. especially the percent of outer primer to inner primer. how can you calculate annealing temp for the four primers. i want to know whether i will use two annealing temp or just one. i want to know how to prevent primer dimer
You asked almost the same question already twice:
https://www.researchgate.net/post/What_are_the_precautions_used_for_tetra-primer_ARMS-PCR
https://www.researchgate.net/post/What_is_the_protocol_of_tetra-primer_ARMS-PCR
Se also:
https://www.researchgate.net/post/What_are_the_precautions_used_for_tetra-primer_ARMS-PCR
https://www.researchgate.net/post/Can_anybody_please_suggest_how_to_optimize_T-ARMS_PCR
I'm have the experience to design tetra ARMS-PCR primers. This all contain 4 of primers (2 pair of primers) and it use for SNP genotyping.
the percent of outer primer to inner primer
I would say that, for the amount of primer that you should add, you need to optimize it first. For example I add just the four primers at 0.2 uM (same concentration) in the final concentration then perform the PCR and Gel electrophoresis to see the expected pcr product and then I estimate the primer concentration via the previous result and you should perform the PCR condition as a gradient as well (the annealing temp using between the four primer)
or you use this formula if the shorter product was to little RI/FO >1 and/or FI/RO >1 or RI/FO
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