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

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>Sam Chiang
 
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='''Single E.coli cell micromanipulation - Fresh sample test'''=
='''Single E.coli cell micromanipulation - Fresh vs. dead sample test'''=


==Objective==
==Objective==
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==Procedures==
==Procedures==
*Sample preparation:
#Grew cell over-night (10mL LB borth + spiked in E.coli K-12 strain glycerol stock)x2 tubes
#Next day. Spin down cells at 3000rpm, RT, for 10min. Remove supernatant. Resuspend in 1mL PBS.
#Transfer into 1.7-mL tubes. Pellet cells at 8000rpm, 5min, RT. Remove supernatant. Resuspend in 1mL PBS.
*Prepare 100uL sample for staining.
#Prepare six 1.5-mL tubes. Transfer 100uL cell suspension into each of tubes.
#Fresh-Ecoli x3 (SYBR-gold, YoYO, DAPI), Dead-Ecoli x3 (SYBR-gold, YoYO, DAPI)
## 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.
#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


 
#Add 2uL of diluted dye into sample suspension (0.5mL). Repeat pipetting slowly ~20X to mix the dye with sample.
 
#Incubate in the Eppendorf rocking incubator at 25C, 400~500rpm for 15min.
 
 
 
 
 
*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.
#Pellet at 5min, 8000 rpm. Remove supernatant. Wash 1X in 1mL PBS(0.2um-filtered).
#Resuspend in 1mL PBS.
#Filter the cell suspension through 5um-filter.
#Pellet at 5min, 8000 rpm. Remove supernatant. Resuspend in 0.5mL PBS.
#Prepare the following glycerol solution in H2O (clean H2O left from GA-sequencer)
##0.1%, 1%, and 10% (v/v)
#Equal volume replacement for adding glycerol in cell suspension.
##Remove 50uL of cell suspension and add 50uL diluted glycerol in the suspension.
##The new glycerol concentration will be 0.01%, 0.1%, and 1%. I also prepare the positive control which has no glycerol added.
#Prepare DAPI working solution (5mg/mL -> 1/5 dilution with H2O to 1mg/mL).
#Add DAPI working solution in the E.coli. Add 2uL dye for each of the tubes. Repeat ipetting slowly 10X~20X to mix the dye with sample.
#Incubate in on the rocking plate/Eppendorf rocking incubator at RT (25C), 400~500rpm for 15min.


==Results==
==Results==
  [[File:042611-noglycerol_bf1.png|400px]]  [[File:042611-noglycerol_dapi200.png|400px]]
  [[File:04-26-11 deadcell-sybrgold-GFP200.png|400px]]  [[File:04-26-11 livecell-sybrgold-gfp400.png|400px]]
  [[File:04-26-11 livecell-sybrgold-a-bf1.png|400px]]
[[File:04-26-11 deadcell-yoyo-GFP200.png|400px]]  [[File:04-26-11 livecell-yoyo-gfp300.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==
*No significant effect on DAPI-staining performance after adding different diluted glycerol
*Using fresh cell (live cells) significantly improve the staining performance. However, the YOYO dye staining is not very successful even on the live cells.
*Surprisingly, the sample added with glycerol showed a much better response to micro-pipetting sucking.
*If we compare the same sample under BF filter, DAPI-staining still showed a better representation rate on of whole population than SYBR-GOLD staining (Some cells stained in SYBR-GOLD are too dim to been seen).
**Our original idea is to use glycerol to slow down the floating mobility of cell during the sucking procedure.
*I am wondering why DNA staining doesn't work on old cells (dead cells)
*Adding glycerol will be added in our standard procedure of sample preparation. However I need to optimize the glycerol concentration with Andy and check which concentration works best for isolation.
#The DNA in dead cell are degraded?
 
#The cell formed some kind of biofilm that avoids the dye to penetrate cell wall?
==Follow up==
*By using the sample prepared today, Andy extracted another four single E.coli cell and deposited them in PCR tubes. I keep them in 4C fridge waiting for validation.


*After discussion with Dr. Zhang, I noticed that we should use non-fixed cell instead of the EtOH-fixed cell for isolation.
==Next==
**I will test if the current DAPI-staining protocol could work on non-fixed E.coli samples.
*Modify the YOYO non-fixed cells staining protocol?
*Combine the current non-fixed cell staining protocol with pre-filtering steps and glycerol adding
*Since I will need to use fresh cell, I will check if there is a way to cyropreserve fresh cell for long term usage.
*Cyropreservation protocol from Bigelow's lab:
#[[Media:Bigelow_lab_Sample_cryopreservation_glycerol.pdf|Sample_cryopreservation_glycerol]]
#[[Media:Bigelow_lab_Sample_cryopreservation_BeTE.pdf|Sample_cryopreservation_BeTE]]

Latest revision as of 16:29, 29 April 2011

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

Objective[edit]

  • 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[edit]

  • 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[edit]

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[edit]

  • Using fresh cell (live cells) significantly improve the staining performance. However, the YOYO dye staining is not very successful even on the live cells.
  • If we compare the same sample under BF filter, DAPI-staining still showed a better representation rate on of whole population than SYBR-GOLD staining (Some cells stained in SYBR-GOLD are too dim to been seen).
  • I am wondering why DNA staining doesn't work on old cells (dead cells)
  1. The DNA in dead cell are degraded?
  2. The cell formed some kind of biofilm that avoids the dye to penetrate cell wall?

Next[edit]

  • Modify the YOYO non-fixed cells staining protocol?
  • Combine the current non-fixed cell staining protocol with pre-filtering steps and glycerol adding
  • Since I will need to use fresh cell, I will check if there is a way to cyropreserve fresh cell for long term usage.
  • Cyropreservation protocol from Bigelow's lab:
  1. Sample_cryopreservation_glycerol
  2. Sample_cryopreservation_BeTE