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-AGGGCATATGCAGGTCACGC-3
) and
RC-R (5
-CCCGTGCGGATCCACCGGCTA-3
CACCCCGCATATGAC-
CACC-3
).
The PCR was performed in thermal cycler, Biocycler TC-S (Bo-
eco, Germany); using 2.5 U Go Taq Flexi DNA polymerase
(Promega). The reaction mixtures (50 l) contained 250 ng of
genomic DNA, 1M of each primer, 200 M of each dNTP,
3 mM MgCl
2
, and 1X Taq buffer. The following conditions
were used for the PCR reaction: the enzyme was added after
an initial denaturation for 1 minute at 98C, followed by 30
cycles [98C for 45 seconds, (66C ribC; 65C parN) for 45
seconds, 72C for 1 minute] and nal elongation at 72C
for 5 minutes.
Plasmid construction
The PCR-amplied products, ribC and parN, were double
digested using Fast digest