Protocols, reagents and checklists for rDNA fluorescent in situ hybridisation in plants
Here you will find:
- A simplified method for fluorescent in situ hybridisation on plant protoplasts based on the one proposed by Heslop-Harrison et al. (1991) with minor modifications by Dr. J. Siroky (as explained in Garcia et al., 2010) and Siljak-Yakovlev et al. (in press) and some adaptations of our own. PDF 1
- A checklist of the basic laboratory equipment, lab tools, consumables and reagents for fluorescent in situ hybridisation in plant protoplasts. PDF 2
- A method for obtaining plant protoplasts, based on Geber and Schweizer (1988) with minor modifications from Siljak-Yakovlev et al. (in press) and some adaptations of our own. PDF 3
- A checklist of the basic laboratory equipment, lab tools, consumables and reagents for obtaining plant protoplasts. PDF 4
- A complete description of the necessary reagents both for in situ and for obtaining plant protoplasts. PDF 5
- A couple of protocols for probe labelling by nick translation, following the manufacturer’s instructions. These include a calculator on the necessary amount of DNA probe for each labelling reaction. XLSX 1
- Information on 18S-25S probe pTa71
- Information on 5S probe pTa794
We wish to thank Dr. Sonja-Siljak Yakovlev (Université Paris Sud – CNRS) and Dr. Jiri Siroky for sharing their knowledge, experience and advice on these techniques, as well as on the assessment and the preparation of these documents.
Geber G & Schweizer D. 1988.
Cytochemical heterochromatin differentiation in Sinapis alba (Cruciferae) using a simple air-drying technique for producing chromosome spreads. Plant Syst Evol 158: 97-106.
Heslop-Harrison LS, Schwarzacher T, Anamthawat-Jonsson K. et al. 1991. In situ hybridization with automated chromosome denaturation. Techniques 3: 109-116.
Gerlach WL & Bedbrook JR.
1979. Cloning and characterization of ribosomal RNA genes from wheat and barley. Nucleic Acids Res. 7:1869-85.
Gerlach WL & Dyer TA.
1980. Sequence organization of the repeating units in the nucleus of wheat which contains 5S rRNA genes. Nucleic Acids Res. 8:4851-65.
Interesting links to FISH protocols, reagents, tips…
Our Nick translation kits and fluorochromes
Nick translation labelling (other protocols)
Links for other fluorescent in situ hybridisation protocols
Reference books or book chapters on Fluorescent in situ Hybridisation
Schwarzacher T, Heslop-Harrison P. 2000. Practical in situ
hybridization. BIOS Scientific Publishers Limited. UK.
Darbi IA, Hewitson TD (ed.). 2006. In situ hybridization protocols (Methods in Molecular Biology). Humana Press Inc. USA.
Zhang P, Friebe B. 2009. FISH on plant chromosomes. In: Fluorescence in situ
hybridization (FISH) – application guide. Pp 365-394. Liehr T (ed). Springer-Verlag Berlin Heidelberg.
Singh RJ. 2003. Plant Cytogenetics. Second Edition. CRC Press USA.
Leitch AR, Schwarzacher T, Jackson D, Leitch IJ. 1994. In situ hybridization: a practical guide. BIOS Scientific Publishers Ltd.