Metabarcoding

16S PCR Reaction

Modified 16S protocol from the Earth Microbiome Project.
PCR reaction mixture
Reagent
Volume
PCR-grade water
10.5 µL
PCR master mix (2x)
12.5 µL
Indexed Primers (should have enough of each indexed primer set for two reactions in case you need to repeat some).
1 µL
Template DNA
1 µL
Total reaction volume
25.0 µL
Temperature
Time, 96-well
Time, 384-well
Repeat
94 °C
3 min
3 min
94 °C
45 s
60 s
x35
50 °C
60 s
60 s
x35
72 °C
90 s
105 s
x35
72 °C
10 min
10 min
4 °C
hold
hold

12S Fish Metabarcoding

PCR reaction mixture
Reagent
Volume
PCR-grade water
6.5 µL
PCR master mix (2x)
12.5 µL
Indexed Primers (should have enough of each indexed primer set for two reactions in case you need to repeat some)
1.0 µL
Template DNA (fish DNA is much less abundant than microbes)
5 µL
Total reaction volume
25.0 µL
12S PCR program uses a lot of cycles to increase signal, which will make things even less quantitative than with fewer cycles.
95°C – 3:00
95°C – 0:20
52°C – 0:20
72°C – 0:20
Repeat x 40
72°C – 5:00
4°C – hold