Sam:LabNotes/Microbiome-new/2009-3-4
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Microwell slide washing and bead loading[edit]
Objective[edit]
- To test if Tween 20 can used to eliminate hydrophobic force on the SU-8 structure
Procedures (Adapted from Jeff's note)[edit]
Slide washing (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
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 1mg bead/200 uL washing buffer (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 2 squares each
- Dry for 10 min (or until the water has fully evaporated)
Results: Microscope image[edit]
- Beads in 1/100 dilution, objective 20X (left); Beads in 1/100 dilution, objective 10X (right)
File:Bead-3-4-09 1-100 2.bmp File:Bead-3-4-09 1-100.bmp
- Beads in 1/200 dilution, objective 20X (left); Beads in 1/200 dilution, objective 20X (right)
File:Bead-3-4-09 1-200 2.bmp File:Bead-3-4-09 1-200.bmp
Discussion[edit]
- The bead could be seen under 20X objective much well than under 10X objective. Most beads congrigate together in the middle of the microwell bottom.
- Magnatic bar can attract the bead on slide but not evenly (since the magnets in the bar are not seperated evenly either). Most of the beads are found in the microwells around the center of the array.
- Using 15 uL of 1/100 bead dilution (3000 bead/15 uL) looks better then using 1/200 dilution.