Sam:LabNotes/Microbiome-new/2009-5-22
Jump to navigation
Jump to search
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