Porphyra purpurea gametophytes were cultured in modified D-11 seawater medium at 15ºC, under a 16 h:8 h LD cycle (Mitman and van der Meer, 1994).
The mtDNA was isolated by agarose gel electrophoresis after cutting the A+T-rich
fraction with SacI. The largest (35 kbp) restriction fragment was the
linearized mtDNA (which has only 1 SacI site), whereas the ptDNA was cut into several
fragments of <15 kbp (M. Reith and J. Munholland, 1993).
Electroeluted mtDNA was physically fragmented by nebulization, size-selected (500-3000 bp),
end-polished and cloned into the SmaI site of pBluescript II KS+ (Stratagene). Recombinant
plasmids containg mtDNA inserts were identified by colony hybridization using the 35-kbp
SacI restriction fragment (see above) as a probe.
Performed by I. Plante and D. St-Louis (Unit).
DNA sequencing was performed by the dideoxy chain termination method (Sanger et al., 1977), using
single-stranded DNA as template and 35S-dATP as a label. Dried acrylamide gels were
autoradiographed and sequences were entered manually.
Performed by D. St-Louis (Unit).