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GeneFISH – combined detection of genes and rRNA in microorganisms
F. List of materials
When you are using this protocol, please cite:
Moraru, C., Lam, P., Fuchs, B.M., Kuypers, M.M.M. and Amann, R. (2010) GeneFISH – an in situ technique for linking gene presence and cell identity in environmental microorganisms. Environ. Microbiol. 12: 3057-3073.
Introduction
A. Sample preparation
A1. Sample fixation
A2. Sample immobilization
A3. Embeding in agaroze
A4. Permeabilization
A5. Inactivation of endogeous peroxidases
B. gene detection
B1. gene probes
B1.1. probe design
B1.2 probe synthesis
B1.3 determination of hybridization conditions for hybridization and washing
B2. gene hybridization
B3. antibody binding
B4. gene CARD and inactivation of HRPs from the gene detection step
C. rRNA detection
C1. rRNA hybridization
C2. rRNA CARD
D. Mounting for microscopy and counterstaining
E. Microscopy
F. List of materials
Materials
Product |
Company |
Cat. No. |
1 M Tris, pH 8.0 |
Ambion |
AM9856 |
20% SDS |
Ambion |
AM9820 |
20x SSC |
Ambion |
AM9765 |
5 M NaCl |
Ambion |
AM9759 |
Anti-Dig-POD Fab fragments |
Roche |
11 207 733 910 |
Blocking Reagent for nucleic acid hybridizations |
Roche |
11096176001 |
Copy Control cDNA, Gene and PCR Cloning Kit |
Epicentre |
CCPCR1CC |
Gene Clean Turbo kit |
Q-Biogene |
1102-600 |
Lysozyme |
AppliChem |
A4972.0010 |
PBS 10x, pH 7.4 |
Ambion |
AM9625 |
PCR Dig Probe Synthesis Kit |
Roche |
11636090910 |
ProLongGold |
Invitrogen |
P36930 |
RNase free DNaseI |
Epicentre |
D9902K |
RNase I (Cloned) 100 U/µl |
Ambion |
AM2294 / AM2295 |
RNaseA |
Sigma |
R4642-10 |
Sheared Salmon Sperm DNA (sheared, 10 mg/ml) |
Ambion |
AM9680 |
SlowFadeGold |
Invitrogen |
S36936 |
Syto9 |
Invitrogen |
S-34854 |
Western Blocking Reagent, Solution |
Roche |
11921673001 |
Yeast RNA (10 mg/ml) |
Ambion |
AM7118 |
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