Sam:LabNotes/Microbiome-new/2009-4-6
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EUB338 prbes FISH labeing on bacteria
Objective
- Generate FISH labeling protocol using non-specific bacteria 16S probes EUB338
- References:
- Manz et al., System Appl Microbiol 1992, 15:593-600
- Kalyuzhnaya et al., Appl Environ Microbiol 2006, 72(6):4293-4301
Preparation
- Dilute probes to 1000 ng/uL with 1X TE buffer
5'FAM-EUB338 (FW=6029.1, 0.32 mg)-> rususpended in 320 uL TE -> 1000 ng/uL 5'Cy5-EUB338 (FW=6024.2, 0.06 mg)-> rususpended in 60 uL TE -> 1000 ng/uL
- 2M NaCL (FW:58.44): 1.17g/10 mL H2O
- 0.1% SDS (FW: 288.38): 0.025g/ 25 mL H2O
- H-buffer: Hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl, 0.01% SDS, probes 25 ng/8uL Hybridization buffer)
H2O 88 uL 2M NaCl 90 uL 2M Tris 2 uL 0.1% SDS 20 uL --------------- 200 uL 200/8*25=625 (ng) probes needed -> Mix H-buffer with 0.625 uL diluted probes (1000 ng/uL)
- W-buffer: Washing buffer (180 mM NaCl, 20 mM Tris, 0.01% SDS, 5 mM EDTA)
x 1 x 5 H2O 158 uL 790 2M NaCl 18 uL 90 2M Tris 2 uL 10 0.1% SDS 20 uL 100 0.5M EDTA 2 uL 10 ------------------------- 200 uL 1000 uL 200/8*25=625 (ng) probes needed -> Mix H-buffer with 0.625 uL diluted probes (1000 ng/uL)