Sam:LabNotes/Microbiome-new/2009-5-22

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FISH on mouse intestinal samples from 5/21/09[edit]

Objectives[edit]

  • Perform FISH on 20uM filtered mouse intestinal content sample
  • Test on different sample amounts
    • 1mL sample- pellet x 2 reactions (Cy5 and FAM labeling)
    • 100uL sample- pellet x 2 reactions (Cy5 and FAM labeling)


Procedures[edit]

  • Hybridization
    • Prepare four wraps for 1.5-mL tubes
    • The fixed cells (10^8) were washed with 0.5 mL PBS(at RT) twice and resuspended in 200 uL of hybridization buffer. Hybridize at 46C for 1.5 hr without shaking. (Use eppendorf isothermo mixer but don't turn on the mixing function).
H-buffer(0.9 M NaCl, 20 mM Tris-HCl, 0.01% SDS)
 
  H2O      88 uL 
  2M NaCl  90 uL
  2M Tris   2 uL
 0.1% SDS  20 uL
 ---------------
          200 uL 


  • Washing
    • Pellet the labeled bacteria by 8000 x g, 3 min. During this time, prewarm the washing buffer at 48C.
    • Remove the supernatant. Resuspended in 500 uL washing buffer and incubate at 48C for 20min without shaking. NOTE: The pellet is difficult to be resuspended into washing buffer. Pipetting the pellet carefully and repeatedly for ~20 times using p1000 tips.
W-buffer (180 mM NaCl, 20 mM Tris, 0.01% SDS, 5 mM EDTA)             
  H2O       395 x2
  2M NaCl    45
  2M Tris     5
 0.1% SDS    50
0.5M EDTA     5
-------------------
            500 uL
  • PBS washing
    • Pellet the washed bacteria by 8000 x g, 3 min. Remove the supernatant.
    • Resuspended the pellet with 1 mL cold PBS. Carefully repeat pipetting for ~20 times. Note: It is suggested to take the tube out of foil for this resuspending step, in which it's easy to see the complete suspension and prevent the pellet loss by sticking to the tips.
    • Pellet labeled bacteria again with 8000 x g, 3 min. Remove the supernatant (pour + p10 tips). NOTE: The pellet of 100uL-sample is too small after 8000xg, 3min centrifuge. Another 14000xg, 3min centrifuge was applied to get the sufficient pellet.
    • Resuspended the pellet with PBS. According to the pellet size. 1mL-sample pellet was resuspended with 200uL PBS. 100uL-sample was resuspended with 50uL PBS. Store samples immediately in dark place at 4C.


Results[edit]

File:SNAP-161124-0001-1ml-fam-bf.jpg File:SNAP-161401-0002-1ml-fam-fitc.jpg File:SNAP-161530-0003-1ml-fam-fitc.jpg
 Left: 1mL-sample-fam-20x-bright field            Center: 1mL-sample-fam-20x-FITC filter                 Right: 1mL-sample-fam-20x-FITC filter

File:SNAP-161752-0004-1ml-cy5-bf.jpg File:SNAP-162239-0005-1ml-cy5-cy5.jpg File:SNAP-162510-0006-1ml-cy5-cy5.jpg
 Left: 1mL-sample-Cy5-20x-bright field            Center: 1mL-sample-Cy5-20x-Cy5 filter                  Right: 1mL-sample-Cy5-20x-Cy5 filter

File:SNAP-163537-0007-100uL-fam-bf.jpg File:SNAP-164127-0008-100uL-fam-fitc.jpg File:SNAP-164514-0010-100uL-fam-fitc-new.jpg
 Left: 100uL-sample-fam-20x-bright field           Center: 100uL-sample-fam-20x-FITC filter             Right: 100uL-sample-fam-20x-FITC filter

File:SNAP-165151-0012-100uL-cy5-bf.jpg File:SNAP-165151-0012-100uL-cy5-cy5.jpg File:SNAP-165725-0013-100uL-cy5-cy5.jpg
 Left: 100uL-sample-Cy5-20x-bright field           Center: 100uL-sample-Cy5-20x-Cy5 filter              Right: 100uL-sample-Cy5-20x-Cy5 filter