Jeff:LabNotes/Microbiome/2009-4-8

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Revision as of 22:22, 10 April 2009 by >Jgole (New page: == '''Bacterial loading into Microwells''' == == Purpose == To test whether the cells are loading properly into the wells by using a blank slide as a posite control. All three stains w...)
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Bacterial loading into Microwells

Purpose

To test whether the cells are loading properly into the wells by using a blank slide as a posite control. All three stains will be used as well as very high concentrations of cells.

Microarray Preparation

  • Wash the array with isopropanol and allow to dry
  • Carefully apply the silicon gasket and ensure a tight seal
  • Add 15 uL of the .1% Tween 20 soluton to each square and allow to dry


Bacterial Dilution and Staining

  • Prepare two different concentrations of bacteria
  1. Begin with E coli of OD=1.0 (10^6 cell/uL)
  • Add 10 uL cell to 90 uL PBS (10^5 cell/uL)
  • Add 20 uL of diluted cells to 20 uL PBS (20000 cell/uL)
  • Do a 1:10 dilution with 1x YOYO, 2x SYBR Green, and a 1:1 mixture of 1x YOYO and 2x SYBR Green for a final conc of 2000 cell/uL
  1. Begin with E coli of OD=1.0 (10^6 cell/uL)
  • Add 10 uL cell to 90 uL PBS (10^5 cell/uL)
  • Do a 1:10 dilution with 1x YOYO, 2x SYBR Green, and a 1:1 mixture of 1x YOYO and 2x SYBR Green for a final conc of 10000 cell/uL
  • Pipette thouroughly to mix
  • Incubate in the dark for 5 min


Loading onto Microwells

Each square has 3600 wells. Ideally, we want less than 1 cell per well. However in this case we want to ensure that the bacteria loads properly into the wells. Therefore, we will load 10000 cells/square.

  • Add 5 uL cells with each concentration/stain to 1 square each (Sam did a duplicate experiment)
  • This will give cell counts of 10000 and 50000 cells per square
  • Pipette to ensure entire square is covered
  • Allow to dry (this could take several minutes)


Results

The following were taken with 20x magnification using a flourescent microscope.

File:1000-bw-1.jpeg 10000 B/W

File:1000-cc-1.jpeg 10000 Color

File:5000-bw-1.jpeg 5000 B/W

File:5000-cc-1.jpeg 5000 color

File:2000-bw-1.jpeg 2000 B/W

File:2000-cc-1.jpeg 2000 color


Discussion

Cells appeared in black and white images, but not flourecsent images. This may be due to photobleaching or letting the cells incubate for an extended period of time. The cells due not appear to be loading into to the wells. Further experiments need to be done on blank slides as a positive control to ensure the stains are working properly.