Has anyone else had difficulty amplifying regions of taxa in the Apocynaceae. Specifically, I am trying to optimize the following primers: trnV-ndhC, rpl32-trnL, 18S (IGS)-18S (ETS), 26S-18S. Has anyone found optimal PCR cycling parameters that allow for good amplification of these regions among taxa WITHIN Apocynaceae?
Dear Kauahi Perez,
Generally apocynaceae family do not create problems during DNA accessing, As you know very well Amplification majorly depends upon the annealing of primers with the template sequences. I suggest you to batter use Gradient PCR ranging from 35 °C - 55 °C. And elongate the annealing and extension time to around 1.30 min.
It will definitely work.
Good Luck...
Dear Kauahi Perez,
PCR depends mainly upon the constituents like buffers, dNTPs, Pol enzyme Primers, etc. However, the annealing of the primers are very crucial. Before proceeding just check the primer sequence status like homology percentage with the target, oligo properties, etc. Be sure that the primers you have selected are specific for you experiment.
You can also optimize PCR by adding MgCl2, DMSO, BSA, etc in the reaction mix and then try it in a lower stringency. All such additives helps to nullify the inhibitors in the reaction.
It should work. Good luck.......
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