Sam:LabNotes/Microbiome-new/2009-4-6: Difference between revisions

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>Sam Chiang
>Sam Chiang
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='''EUB338 prbes FISH labeing on bacteria'''=
='''EUB338 prbes dilution and FISH labeing on bacteria'''=


==Objective==
==Objective==

Revision as of 19:43, 29 June 2009

EUB338 prbes dilution and 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, wrapped with foil and keep in 4C
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 concentration: 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)

Procedures

  • Bacteria cells washing and adjusting
    • Pallet by 4000 rpm, 5 min
    • Resuspended at 0.5 mL PBS
    • Adjust to OD600 = 1.0 (10^6 CFU/uL) using Nanodrop
  • Fixation (Start with 200 uL diluted (10^6 CFU/uL)E.coli (2x10^8 cells) in a 1.5-mL tube)
    • Pellet the bacteria by 4000 rpm, 5 min
    • Resuspended the bacteria in 100% ethanol.
    • Incubate at RT on rocking plate (speed=3) for 3 hr
    • All fixed cell are stored in -20C
  • Hybridization
    • Pre-warmed the hybridization buffer (200 uL) and washing buffer (500 uL) at 46-48C
    • The fixed cells (10^8) were washed with 0.5 mL PBS twice and resuspended in 200 uL of hybridization buffer. Hybridize for 1.5 hr at 46C, 300 rpm using eppendorf isothermo mixer.
    • Pellet the labeled bacteria by 4000 rpm, 5 min. Remove the supernatant. Resuspended in 500 uL washing buffer and incubate at 48C, 300 rpm for 20 min.
    • Pellet the labeled bacteria by 4000 rpm, 5 min. Remove the supernatant. Resuspended in 1 mL cold PBS. Wrapped in foil and stored in 4C.


Results

  File:040709-FAM 20X-BW-1.jpg File:040709-FAM 20X-CC-1.jpg
  FAM 20X-phase contrast                                           FAM 20X-FITC
 
 
 
  File:040709-CY5 20X-BW-1.jpg File:040709-CY5 20X-CC-1.jpg
  Cy5 20X-phase contrast                                           Cy5 20X-FITC


Discussion

  • The results of FAM dye showed possible FISH labeling on bacteria.
  • Cy5 dyes are far-red dye which needs to use 675/50BP filter. Unfortunetely no suitable filter is available in Dr. Shyne's lab.
  • The crystal structure seen on phase contrast picture is due to the crystalization of salts in left from washing buffer. More PBS washing should be applied in the last step.
  • The 300 rpm agitation in the washing step might also destroy the bound probes on bacteria DNA, which should be avoid in the next test.