Sam:LabNotes/Micro-manipulation/2011-4-15: Difference between revisions

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>Sam Chiang
>Sam Chiang
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*Test if I can do TryphenBlue + DAPI staining at the same time.
*Test if I can do TryphenBlue + DAPI staining at the same time.
*Confirm the effect the final-washing.
*Confirm the effect the final-washing.
*Test the effect of glycerol on cell floating mobility.
*Test the effect of glycerol on cell floating mobility. Confirm the background reflection problem.

Revision as of 20:02, 16 April 2011

Single E.coli cell micromanipulation test on 04-15-'11

Objective

  • Further improve the DAPI staining protocol

Experiment design

  • Test the following conditions:
  1. The effect of filtering the fixed E.coli (by 5um-filter)
  2. Washing and resuspend in PBTw to remove background.
  3. Using higher amount of DAPI dye.

Procedures

Test of pre-filtering the EtOH-fixed samples

  • Starting with EtOH fixed cell (stored in -80C) x2
This cell are about 100uL of OD600=1.0 E.coli, fixed in 500uL 100% EtOH for 3hr.
  1. Pellet at 5min, 8000 rpm. Wash 1X in 1mL PBS(0.2um-filtered).
    1. One of the samples was filtered through 5um-filter, and labeled as "F".
    2. The other one was labeled as "R" following the regular procedure.
  2. Resuspend in 0.5 mL PBS(0.2um-filtered). Transfer sample in a 1.7mL amber tube.
  3. Prepare DAPI working solution (5mg/mL -> 1/5 dilution with H2O to 1mg/mL).
  4. Add DAPI working solution in the E.coli
    1. Add 1uL dye for "R" tube. Add 4uL dye for the "F" tube.
  5. Incubate in on the rocking plate/Eppendorf rocking incubator at RT (25C), 400rpm for 15min.

Test of adding final washing with PBTw

  • Started from the DAPI-stained sample on 4-14-'11
  1. Separate the sample into two tubes(250uL/tube)
  2. Pellet stained cells at 8000rpm, 5min, RT.
  3. Prepare the PBTw (PBS containing 0.1% Tween20) by adding 40uL Tween20 into 40mL PBS(0.2um filtered).
  4. Pick up one of the samples. Using 1mL PBTw to wash cells twice. Resuspend the cell pellet in 250uL PBTw. Label the tube as "washed".
  5. Label the other tube as "non-washed".

Results

Comparisons of different conditions on DAPI-stained E.coli

File:041511 R nofilter-dapi200 annotatede.png File:041511 F filter-dapi200 annotatede.png
 
File:041511 Unwashed-dapi200 annotated.png File:041511 PBTw washed-dapi200 annotated.png

Discussion

  • Effect of Filter:
    • It's a good idea to filter the fixed E.coli sample before DAPI staining. The fixed-E.coli suspsend in 1mL PBS can still pass through the 5um filter.
    • The picture of filtered sample showed much uniformed particles than non filtered samples.
    • Although it looks like non filtered sample is brighter. However, these bright particles mostly stained large debris. The small particle (probably read E.coli cells) are still dim.
  • Effect of higher dye concentration:
    • Although not seen in pictures, it seems like 4uL dye stained sample("F") showed a better signal/noise than 1uL dye staining sample ("R") if we compared under the same microscope setting.
    • It's effective but result is not significant.
  • Effect of final washing:
    • Add finally washing using PBTw affect the signal intensity significantly, and I can't tell if the background is decreased or not in the washed sample.
    • I am planing to confirm this again in the next experiment.

Next

  • Test if I can do TryphenBlue + DAPI staining at the same time.
  • Confirm the effect the final-washing.
  • Test the effect of glycerol on cell floating mobility. Confirm the background reflection problem.