I wanted to look at 5 genes expressed by bacteria under different conditions such as incubated with antimicrobial peptide indolicidin. For the housekeeping, I am using 16S RNA. I have tested the primers for each and they all work at 60 degrees. Do I do the efficiency calculations and 10 fold dilutions using the cDNA from my vehicle treated bacteria (with the housekeeping primer) or using cDNA from bacteria treated with the indolicidin and the housekeeping primer? for the rest of the run, Do I put everything else with undiluted cDNA from every condition with every primer in triplicate?
Thanks for your help.
Hello Nahom
for primer efficiency based on a standard curve I would recommend making a pool from cDNA derived from treated and untreated cDNA and subject that pool to a dilution
I would perform a standard curve for both GOI primer and housekeeping primer using this titrated cDNA pool and make sure the efficiency values are within 5% of each other
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