Jeff:LabNotes/Microbiome/2009-3-5

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Revision as of 01:36, 8 March 2009 by >Jgole (New page: == '''Bead Loading''' == '''Purpose''' *To repeat the cleaning and beading loading procedure to see if similar results were obtained. *To load E.coli into the wells and observe the loadin...)
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Bead Loading[edit]

Purpose

  • To repeat the cleaning and beading loading procedure to see if similar results were obtained.
  • To load E.coli into the wells and observe the loading pattern.

Microarray cleaning[edit]

  1. Spray microarray with isopropanol
  2. Wipe carefully with a kimwipe and allow to dry

Note: You can only wash once with isopropanol. Additional washing causes the wells to fall off


Tween 20/PBS washing[edit]

  1. Add 20 uL of Tween 20 to 180 uL of H20 (10% dilution).
  2. Add 19.2 mL of PBS to the solution for a final dilution of .1% Tween 20.
  3. Shake until a soapy consistency is observed
  4. Gently spray 5 ml of the solution over the microarrays
  5. Wipe carefully with a kimwipe and allow to dry


Silicon Gasket Application[edit]

  1. Remove the protective covering from one side of the gasket and place in the desired position on the microarray
  2. Firmly press down until the squares are sealed
  3. Remove the protective covering from the other side and press down, ensuring a tight seal


Bead Loading[edit]

  1. Dilute the beads so that the concentration is 1ug bead/200 uL H2O (6-7 x 10^7 beads in solution)
  2. Do another 1:200 dilution (1500 beads) and a 1:100 dilution (3000 beads)
  3. Place the microarray over a magnetic bar
  4. Add 15 uL beads from each dilution to 3 squares each
  5. Dry for 10 min (or until the water has fully evaporated)


E.Coli Loading[edit]

  • Sam stained and loaded the E.coli. Look at his notes for the detailed procedure.