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I am doing an experiment to amplify the 16S rRNA gene from bacteria present in gut contents of fish. After extracting DNA, I perform one-step PCR with universal bacterial primers (27F, 1492R) and I see a strong band of expected length (approx 1500bp). My DNA extraction readings range from 50ng/ul in some samples, to 150ng/ul in others, as measured with Nanodrop.

Does this band represent all of the 16S genes present in the sample? Thus, does this band represent 16S genes from all of the bacterial species present in the sample?

Do I need to perform a microbiome profiling PCR to differentiate and identify all of these species?

Thank you.

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The band represents all of the 1500 bp sequences able to be amplified by the primers you chose - no more, no less. Amplification efficiency will likely vary between species/sequences, and while there's a decent chance that your conjecture is correct, further validation will be required to support your assumption. –  MattDMo Nov 26 '13 at 15:19
    
Thank you MattDMo. So therefore, through DGGE or pyrosequencing of smaller hyper-variable regions of this gene, perhaps all of the different species present in a given DNA sample could be elucidated and identified? –  James Nov 27 '13 at 13:32
    
James - yes, something along those lines would be necessary. You've amplified enough of each target (hopefully), so now you can move on to identifying and classifying the sequences it contains. Good luck! –  MattDMo Nov 27 '13 at 14:39

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