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

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>Sam Chiang
>Sam Chiang
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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                    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.
 
 
 
 
 
 
*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.
#Incubate in on the rocking plate/Eppendorf rocking incubator at RT (25C), 400~500rpm for 15min.



Revision as of 01:15, 27 April 2011

Single E.coli cell micromanipulation - Fresh 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                    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 on the rocking plate/Eppendorf rocking incubator at RT (25C), 400~500rpm for 15min.

Results

File:042611-noglycerol bf1.png  File:042611-noglycerol dapi200.png

Discussion

Follow up