Sam:LabNotes/Microbiome-new/2009-4-6

From ZhangLabWiki
Revision as of 18:14, 7 April 2009 by >Sam Chiang (→‎Objective)
Jump to navigation Jump to search

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)