Sam:LabNotes/Micro-manipulation/2011-4-26: Difference between revisions

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>Sam Chiang
>Sam Chiang
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  [[File:04-26-11 livecell-yoyo-bf1.png|400px]]
  [[File:04-26-11 livecell-yoyo-bf1.png|400px]]
[[File:04-26-11 deadcell-dapi-DAPI200.png|400px]]  [[File:04-26-11 livecell-dapi-dapi200.png|400px]]


==Discussion==
==Discussion==


==Follow up==
==Follow up==

Revision as of 17:17, 27 April 2011

Single E.coli cell micromanipulation - Fresh vs. dead sample test

Objective

  • I compare DAPI-staining performance on fixed(EtOH) and non-fixed E.coli cells(using the old E.coli sample stored from 03-10-'10 in 4C). The fixed cell still give me bright DAPI signal, but the non-fixed E.coli cell showed very dim single as previous tests I did.
  • I start doubting if the E.coli samples in the non-fixed samples are too old or the DNA were degraded in these cells.
    • When bacteria are dying the DNA will be degraded slowly, however I thought the degradation is too slow to be regarded as a concern.
  • I grew the fresh E.coli cell on 04-25-'11, and will compare fresh cells with old cells on DNA dye staining again.

Procedures

  • Sample preparation:
  1. Grew cell over-night (10mL LB borth + spiked in E.coli K-12 strain glycerol stock)x2 tubes
  2. Next day. Spin down cells at 3000rpm, RT, for 10min. Remove supernatant. Resuspend in 1mL PBS.
  3. Transfer into 1.7-mL tubes. Pellet cells at 8000rpm, 5min, RT. Remove supernatant. Resuspend in 1mL PBS.
  • Prepare 100uL sample for staining.
  1. Prepare six 1.5-mL tubes. Transfer 100uL cell suspension into each of tubes.
  2. Fresh-Ecoli x3 (SYBR-gold, YoYO, DAPI), Dead-Ecoli x3 (SYBR-gold, YoYO, DAPI)
    1. Dead E.coli were from the same source as on 4-21-'11's test. The E.coli cells cultured on 3-10-'10, stored in 4C.
  3. Pellet cells at 8000rpm, 5min, RT. Remove supernatant, resuspend in 0.5mL PBS.
  • Prepare following DNA staining dye
SYBR-GOLD(10,000X, stock sol.) -> 1uL stock sol diluted in 9uL TE -> 1,000X
YOYO-1   (10,000X, stock sol.) -> 1uL stock sol diluted in 9uL TE -> 1,000X
DAPI     (pre-diluted  5mg/mL) -> 1/5 dilution with H2O -> 1mg/mL
  1. Add 2uL of diluted dye into sample suspension (0.5mL). Repeat pipetting slowly ~20X to mix the dye with sample.
  2. Incubate in the Eppendorf rocking incubator at 25C, 400~500rpm for 15min.

Results

File:04-26-11 deadcell-sybrgold-GFP200.png  File:04-26-11 livecell-sybrgold-gfp400.png

File:04-26-11 livecell-sybrgold-a-bf1.png

File:04-26-11 deadcell-yoyo-GFP200.png  File:04-26-11 livecell-yoyo-gfp300.png

File:04-26-11 livecell-yoyo-bf1.png

File:04-26-11 deadcell-dapi-DAPI200.png  File:04-26-11 livecell-dapi-dapi200.png

Discussion

Follow up