ClaI R6551

Protocol tips

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	Approximately 1 μg of genomic DNA from mycobacterial strains was digested overnight at 37°C with 5 U of Cla I (Promega, Madison, USA), and 10 U of Sal I. Restriction fragments were separated overnight by electrophoresis on 0.8% agarose gel and then transferred to a nylon membrane by vacuum blotting using a Milliblot-V transfer system (Millipore, Bedford, MA, USA).

Protocol tips
Approximately 1 μg of genomic DNA from mycobacterial strains was digested overnight at 37°C with 5 U of Cla I (Promega, Madison, USA), and 10 U of Sal I. Restriction fragments were separated overnight by electrophoresis on 0.8% agarose gel and then transferred to a nylon membrane by vacuum blotting using a Milliblot-V transfer system (Millipore, Bedford, MA, USA).

Publication protocol

"Southern blot analysis was performed to check for
the PiD1 and PiD2 deletions in various strains of the
M. tuberculosis complex. Probes were amplified by
PCR from M. bovis AF2122/97 or M. tuberculosis
H37Rv genomic DNA as described above. Amplification products were radiolabelled with [32P] dCTP using
the Prime-a-Gene kit (Promega, Madison, USA) as
specified by the manufacturer.
Approximately 1 lg of genomic DNA from mycobacterial strains was digested overnight at 37 C with
5 U of ClaI (Promega, Madison, USA), and 10 U of
SalI. Restriction fragments were separated overnight
by electrophoresis on 0.8% agarose gel and then transferred to a nylon membrane by vacuum blotting using
a Milliblot-V transfer system (Millipore, Bedford,
MA, USA). The DNA was fixed to the membranes by
exposure to UV (0.120 J/cm2
). Membranes were prehybridised and hybridised in a solution containing 1%
SDS (sodium dodecyl sulfate), 2.5· SSPE (1· SSPE:
10 mM NaH2PO4, 0.18 M NaCl, 1 mM EDTA, pH
7.4, 0.01% sodium pyrophosphate and 0.1% polyanetholsulfonic acid). Prehybridisation was carried out for
3 h at 65 C, with hybridisation performed overnight
at 65 C. Membranes were washed twice for 5 min in
1· SSC, 0.1% SDS at room temperature. Membranes
were exposed to X-ray film at
70 C for 2 days and
the autoradiographs were then developed."

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Manufacturer protocol

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