|
Details |
Final
concentration
of PCR
components
in 50 μl
total
volume:
-
18S Primer Fwd-A: 0.5 μM (Medlin et al., 1988)
-
18S Primer Rev-B: 0.5 μM (Medlin et al., 1988)
-
Promega 10X buffer B: 1X
-
Promega MgCl2: 2.5 mM
-
Promega DNTP (5 mM ea.): 250 μM
-
BSA (Sigma A-7030): 300 ng/μl (Kirchman et al., 2001)
-
Promega Taq in buffer B: 2.5 units
-
DNA template: ~10 ng
Thermal
protocol:
95°C 2 min.
(1x), 95°C
30 sec: 55°C
30 sec: 72°C
2 min (35x),
72°C 7 min
(1x), 4°C
(hold)
References:
Medlin,
L. K., H. J.
Elwood, S.
Stickel, and
M. L. Sogin.
1988. The
characterization
of
enzymatically
amplified
eukaryotic
16S-like
rRNA-coding
regions.
Gene 71:491-499.
Kirchman,
D. L., L. Y.
Yu, B. M.
Fuchs, and
R. Amann.
(2001)
Structure of
bacterial
communities
in aquatic
systems as
revealed by
filter PCR.
Aquatic
Microbial
Ecology
26:13-22.
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