Table 3 Master mix constituents and PCR conditions
16S PCR | ||
Master mix preparation (for single reaction) | ||
Reagent | Volume/μL | |
Platinum PCR SuperMix High Fidelity (Invitrogen) | 20 | |
27F/ 533F (10 μM) | 1 | |
1492R/ 1100R (10 μM) | 1 | |
DNA | 5 | |
PCR conditions | ||
Temperature/oC | Time | No. of cycles |
94 | 2 min | 1 |
94 55 68 | 30 s 30 s 2 min | 30 |
68 | 7 min | 1 |
12 | Hold | / |
ITS/ human beta-globin PCR | ||
Master mix preparation (for single reaction) | ||
Reagent | Volume/μL | |
10X PCR Buffer II (Applied Biosystems) | 2.5 | |
25 mM MgCl2 (Applied Biosystems) | 1.5 | |
AmpliTaq Gold DNA Polymerase (Applied Biosystems) | 0.5 | |
10 mM dNTPs (Roche) | 0.5 | |
Molecular grade water (Sigma) | 10 | |
ITS5/ BG-F (10 μM) | 2.5 | |
ITS4/ BG-R (10 μM) | 2.5 | |
DNA | 5 | |
PCR conditions | ||
Temperature/oC | Time | No. of cycles |
95 | 10 min | 1 |
95 | 15 s | |
52 | 30 s | 50 |
72 | 90 s | |
72 | 7 | 1 |
12 | Hold | / |