Sam:LabNotes/Microbiome-new/2009-5-22: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Sam Chiang
(New page: ='''FISH on mouse intestinal samples from 5/21/09'''= ==Objectives== *Perform FISH on 20uM filtered mouse intestinal content sample *Test on different sample amounts **1mL sample- pellet ...)
 
>Sam Chiang
Line 36: Line 36:
             500 uL
             500 uL


 
*PBS washing
**Pellet the washed bacteria by 8000 x g, 3 min. Remove the supernatant.  
**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.
**Resuspended the pellet with 1 mL cold PBS. Carefully repeat pipetting for ~20 times.
**Pellet labeled bacteria again with 8000 x g, 3 min. Remove the supernatant (pour + p10 tips).  
**Pellet labeled bacteria again with 8000 x g, 3 min. Remove the supernatant (pour + p10 tips).  
**Resuspended the pellet with 100 uL PBS in each tube. Store samples immediately in dark place at 4C.
**Resuspended the pellet with 100 uL PBS in each tube. Store samples immediately in dark place at 4C.

Revision as of 08:20, 23 May 2009

FISH on mouse intestinal samples from 5/21/09

Objectives

  • 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

  • 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.
    • Pellet labeled bacteria again with 8000 x g, 3 min. Remove the supernatant (pour + p10 tips).
    • Resuspended the pellet with 100 uL PBS in each tube. Store samples immediately in dark place at 4C.