PHYSCOmanualver. 1.4
Yuji
Hiwatashi, Tomomichi Fujita, Yoshikatsu Sato,
Takako
Tanahashi, Tomoaki Nishiyama, Keiko Sakakibara,
Rumiko
Kofuji, Naoki Aono, Naomi Sumikawa,
Takashi
Murata, and Mitsuyasu Hasebe
National
Institute for Basic Biology, Okazaki, Japan
2. Cultivation
2.2
Culture and storage of protonemata and gametophores
2.3
Induction of antheridia, archegonia and sporophytes
2.4
Sporangium collection and storage
2.6
Crosses of mutants defective in the sporophyte formation
2.7
Culture in a thin layer of agar medium spread in a glass-bottom dish
3. Observation
3.1
Observation of protonemata and gametophores
3.2
Observation of protonema colony morphology
3.3
Observation of mitosis in a protonemal cell
3.5
Gravitropic response on caulonemata
3.6
Observation of antheridia and archegonia
3.8
Observation of archegonia and embryos by confocal laser scanning microscopy
3.9
DAPI, Hoechst33342, and PI Staining of protonemata
3.10
Visualization of cell wall with calcofluor
3.12
Microtubule by indirect immunofluorescence microscopy
3.14
Light and electron microscopy of archegonia embedded with epoxy resin
4. Gene isolation
4.2
Simple DNA extraction for PCR screening
4.3
Isolation of genomic fragment by TAIL-PCR
4.7
Miniprotoplast preparation for BAC library
5. DNA and RNA gel-blot
and RT-PCR analyses
7. Effects of drugs on
development
9. Transformation
9.1
PEG-mediated transformation
9.2
Transient expression of a foreign gene using particle bombardment
10. Expression analyses
10.0
Retrieving Genomic Sequences for Gene Targeting Using the Available Web-Based
Browsers
10.1
Insertion of GUS (GFP, YFP) genes to form a fusion protein with a target gene
10.2
Cellular localization of a protein fused with a fluorescent protein (GFP,
YFP, RFP)
11. Loss of function
analyses
12. Over and ectopic
exression
12.1
Constitutive overexpression of cDNA or cDNA with tags
14. Physcomitrella
genome project