For samples from each animal and at each time-point, the 16S rRNA gene was amplified from extracted DNA using the composite forward primer 5′-GCCTCCCTCGCGCCATCAG NNNNCTGCTGCCTYCCGTA-3′ where the underlined sequence is that of 454 Life Sciences® primer A and in italics is the broad range bacterial primer BSR357. The reverse primer was 5′-GCCTTGCCAGCCCGCTCAG NNNN AGAGTTTGATCCTGGCTCAG-′3, where the underlined sequence is that of 454 Life Sciences® primer B and in italics is the broad range bacterial primer BSF8. The NNNN designates the unique four base bar code used to tag each PCR product. Reaction conditions were as follows: 5.0 μl 10× PCR buffer II (Applied Biosystems, Foster City, CA), 3.0 μl MgCl2 (25 mM; Applied Biosystems), 2.5 μl Triton X-100 (1%), 2.0 μl deoxyribonucleoside triphosphates (10 mM), 1.0 μl forward primer and 1.0 μl reverse primer (20 pmol/μl each) and 0.5 μl AmpliTaq® DNA polymerase (5U/μl; Applied Biosystems) and 100 ng of template DNA in a total reaction volume of 50 μl. Reactions were run in a GeneAmp® PCR System 9700 cycler (Applied Biosystems) using the following cycling parameters: 5 minutes denaturing at 95 °C followed by 20 cycles of 30 secs at 95 °C (denaturing), 30 secs at 56 °C (annealing) and 90 secs at 72 °C (elongation), with a final extension at 72 °C for 7 minutes. Four independent PCR reactions were performed for each sample along with a no template negative control.
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