Dynamo sybr green qpcr master mix

DNA extracted from intestinal content samples (faeces) was subjected to RT-PCR of 16S rRNA genes using 0.2 mM concentrations of the broad-range bacterial 16S primers 563F (5’-AYTGGGYDTAAAGNG-3’) and 926Rb (5’-CCGTCAATTYHTTTRAGT-3’).
Standard curves were generated by serial dilution of a linearized vector (Invitrogen TOPO pcr2.1 TA vector^AMP amplified into DH5α bacteria) containing the V4 and V5 regions from the E.coli rRNA gene (6 serial dilutions starting from 100 × 10⁶ copies/μl). DyNAmo SYBR Green qPCR Master Mix (Fisher# F-410L) was used to set up the reactions. The cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 52°C for 30s, and 72°C for 1min.

DNA extracted from fecal pellets or cecal content was subjected to quantitative PCR using the following custom CBBP member-specific primers.
BS1 (B. sartorii): fw: 5’-ATCCAACCTGCCTTATACTCCC-3’; rev: 5’-TACCGAAGTTCTTTAATCACGAGA-3’; LanB1 (B. producta): fw: 5’-ACTGCCGTTTTTACCTGTGG-3’; rev: 5’-CCATGGGAACTCAGACCACT-3’; CB4 (C. bolteae): fw: 5’-CATCCTTTTGACCGGCGT-3’; rev: 5’-GATTTGCTCCACATCACTGTC-3’; PD3 (P. distasonis): fw: 5’-GGGATGAAGGTTCTATGGATCG-3’; rev: 5’- CGCTGACTTAAAAGGCCGC-3’.
Standard curves were generated by amplification of DNA extracted from pure cultures (48h on Columbia Blood Agar plates, BD) of the respective bacteria (six serial 1:10 dilutions, from 3.3 ng/μl, 3 μl, ~ 10 ng total DNA). A control consisting of equal amounts DNA from the 4 members was always added to confirm accuracy of the quantification. DyNAmo SYBR Green qPCR Master Mix (Fisher# F-410L) was used to set up the reactions. Reactions were run on a StepOne Plus RT PCR system (Applied Biosystems). The cycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 52°C for 30s, and 72°C for 1min.