Jeff:LabNotes/Microbiome/2009-3-5
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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]
- Spray microarray with isopropanol
- 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]
- Add 20 uL of Tween 20 to 180 uL of H20 (10% dilution).
- Add 19.2 mL of PBS to the solution for a final dilution of .1% Tween 20.
- Shake until a soapy consistency is observed
- Gently spray 5 ml of the solution over the microarrays
- Wipe carefully with a kimwipe and allow to dry
Silicon Gasket Application[edit]
- Remove the protective covering from one side of the gasket and place in the desired position on the microarray
- Firmly press down until the squares are sealed
- Remove the protective covering from the other side and press down, ensuring a tight seal
Bead Loading[edit]
- Dilute the beads so that the concentration is 1ug bead/200 uL H2O (6-7 x 10^7 beads in solution)
- Do another 1:200 dilution (1500 beads) and a 1:100 dilution (3000 beads)
- Place the microarray over a magnetic bar
- Add 15 uL beads from each dilution to 3 squares each
- 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.