Sam:LabNotes/Microbione/2009-3-6: Difference between revisions

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>Sam Chiang
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>Sam Chiang
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*Prepare four 1.5-mL tube, labeled and wrapped with foil.
*Prepare four 1.5-mL tube, labeled and wrapped with foil.
*Set up as following exp design:
*Set up as following exp design:
                    No-treat 100uL 200uL 400uL
              No-treat 100uL 200uL 400uL
----------------------------------------------
            ---------------------------
H2O                   400      0    0    0  uL
  H2O       400      0    0    0  uL
2X SYBR               0     100  200  400  uL
  2X SYBR     0   100  200  400  uL
E. colu suspension     1       1    1    1  uL
  E. coli     1     1    1    1  uL
----------------------------------------------
            ---------------------------


*Mix by gently pipetting (p1000 pipettor). Incubate at dark place for 15 min. Agitate by vortexing for every 3~4 min.
*Mix by gently pipetting (p1000 pipettor). Incubate at dark place for 15 min. Agitate by vortexing for every 3~4 min.
Line 32: Line 32:
*load 1 uL sample from each tube on the slide and place with cover slips.
*load 1 uL sample from each tube on the slide and place with cover slips.
*Keep the slide in dark storage box for microscope visualizing.
*Keep the slide in dark storage box for microscope visualizing.
==Results==

Revision as of 23:51, 6 March 2009

=Bacteria labeling using SYBR gree

Objective


Samples & Materials

  • Bacteria: E.coli suspension in PBS (OD600=0.913 -> around 10^9 CFU/mL = 10^6 CFU/uL)
  • SYBR 2X: Diluted from SYBR 50X stock solution


Procedures

  • Dilute SYBR 50X to 2X with RNAse-free H2O: 40 uL of 50X SYBR + 960 uL H2O -> 2X SYBR, 1000 uL.
  • Prepare four 1.5-mL tube, labeled and wrapped with foil.
  • Set up as following exp design:
             No-treat 100uL 200uL 400uL
            ---------------------------
  H2O       400      0     0     0   uL
  2X SYBR     0    100   200   400   uL
  E. coli     1      1     1     1   uL
            ---------------------------
  • Mix by gently pipetting (p1000 pipettor). Incubate at dark place for 15 min. Agitate by vortexing for every 3~4 min.
  • After incubation, add H2O to 1000 mL for each tube, the new concentration for E. coli -> 1000 CFU/uL)
  • Prepare two microscopy slide with labeling.
  • load 1 uL sample from each tube on the slide and place with cover slips.
  • Keep the slide in dark storage box for microscope visualizing.

Results