Table 1. Primers, 5′ nuclease probes, and characteristics of the qPCR assays run on the MFB.
SAR11 16S rRNA gene | Marine crenarchaeal 16S rRNA gene | Synechococcus rbcL | ||
qPCR primers and probes∧ | Forward Primer | SAR11-433f CTCTTTCGTCGGGGAAGAAA (500 nM) | ARCHG1-334F AGATGGGTACTGAGACACGGAC (1000 nM) | RbcLfCAGACCACCCTCGGCTACAT (333 nM) |
Reverse Primer | SAR11-588R CCACCTACGWGCTCTTAAGC (1500 nM) | ARCHG1-554R CTGTAGGCCCAATAATCATCCT (500 nM) | RbcLrCCCAGTCCTGATCGAAGAAGTT (333 nM) | |
5′ Nuclease Probe | TM519bR TTACCGCGGCTGCTGGCAC (200 nM) | TM519aRTTACCGCGGCGGCTGGCAC (400 nM) | TMrbcLTTCGTTCCTGAAGATCGCAGCCG (200 nM) | |
Assay Reference | [6] | [5] | [44] | |
MFB | Δ fluorescence* | 1000 | 1400 | 500 |
NTC | No Amplification | No Amplification | No Amplification | |
Slope | −3.8646 | −3.5654 | −3.1259 | |
Intercept | 40.269 | 41.495 | 41.862 | |
R2 | 0.9949 | 0.9872 | 0.9473 | |
PCR efficiency | 0.81 | 0.91 | 1.09 | |
ABI7700 | PCR efficiency | 0.97 | 0.98 | 0.97 |
final concentration in 30 µL reaction.
*at the end of a qPCR reaction run with the 104 standard dilution.
PCR efficiency = 10(−1/slope)−1.