Sam:LabNotes/Microbiome-new/2009-4-6: Difference between revisions
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>Sam Chiang (New page: ='''EUB338 prbes FISH labeing on bacteria'''= ==Objective== *Generate FISH labeling protocol using non-specific bacteria 16S probes EUB338 ==Preparation== *Dilute probes to 1000 ng/uL w...) |
>Sam Chiang |
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(17 intermediate revisions by the same user not shown) | |||
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='''EUB338 prbes FISH labeing on bacteria'''= | ='''EUB338 prbes dilution and FISH labeing on bacteria'''= | ||
==Objective== | ==Objective== | ||
*Generate FISH labeling protocol using non-specific bacteria 16S probes EUB338 | *Generate FISH labeling protocol using non-specific bacteria 16S probes EUB338 | ||
*References: | |||
**Manz et al., System Appl Microbiol 1992, 15:593-600 | |||
**Daims et al, System Appl Microbiol 1999, 22:434-444 | |||
**Kalyuzhnaya et al., Appl Environ Microbiol 2006, 72(6):4293-4301 | |||
==Preparation== | ==Preparation== | ||
*Dilute probes to 1000 ng/uL with 1X TE buffer | *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'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 | 5'Cy5-EUB338 (FW=6024.2, 0.06 mg)-> rususpended in 60 uL TE -> 1000 ng/uL | ||
*2M | *2M NaCl (FW:58.44): 1.17g/10 mL H2O | ||
*0.1% SDS (FW: 288.38): 0.025g/ 25 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) | *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 | H2O 88 uL | ||
2M NaCl 90 uL | 2M NaCl 90 uL | ||
Line 32: | Line 35: | ||
------------------------- | ------------------------- | ||
200 uL 1000 uL | 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== | |||
[[Image:040709-FAM 20X-BW-1.jpg|500px]] [[Image:040709-FAM 20X-CC-1.jpg|500px]] | |||
'''FAM 20X-phase contrast FAM 20X-FITC''' | |||
[[Image:040709-CY5 20X-BW-1.jpg|500px]] [[Image:040709-CY5 20X-CC-1.jpg|500px]] | |||
'''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. |
Latest revision as of 07:49, 27 June 2010
EUB338 prbes dilution and FISH labeing on bacteria[edit]
Objective[edit]
- Generate FISH labeling protocol using non-specific bacteria 16S probes EUB338
- References:
- Manz et al., System Appl Microbiol 1992, 15:593-600
- Daims et al, System Appl Microbiol 1999, 22:434-444
- Kalyuzhnaya et al., Appl Environ Microbiol 2006, 72(6):4293-4301
Preparation[edit]
- 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[edit]
- 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[edit]
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[edit]
- 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.