Sam:LabNotes/Microbione/2009-2-12: Difference between revisions
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*Start from the diluted and immobilized bead (with FITC labeled probes). Concentration: 1 mg/200 uL. | *Start from the diluted and immobilized bead (with FITC labeled probes). Concentration: 1 mg/200 uL. | ||
** The original bead info (Invitrogen): Dynabead M-280 | ** The original bead info (Invitrogen): [[Media:Dynabead M-280.pdf|Dynabead M-280]] | ||
** 10 mg (6-7 x 10^8 beads) -> 1 mg = 6-7 x 10^7 beads | ** 10 mg (6-7 x 10^8 beads) -> 1 mg = 6-7 x 10^7 beads | ||
Line 66: | Line 66: | ||
*Air day the slide on hume hood for until the diluent is fully evaporated (More than 10 min will be needed to dry up) | *Air day the slide on hume hood for until the diluent is fully evaporated (More than 10 min will be needed to dry up) | ||
** It may be possible to dry on on magnetic hot plate at 60 degree | ** It may be possible to dry on on magnetic hot plate at 60 degree | ||
==Microscope images== | ==Microscope images== |
Revision as of 22:26, 14 February 2009
Test of Microwell Slide Washing and Microbeads Loading
Objective
- Clean and sterilize the slides before beads loading.
- Load the bead on slides and evaluting the slide quality, bead arrangement under microscope.
Slide washing
Reagents
- 1N HCL (diluted from 12N to 1N)
- Acetone
- Ethanol (200-proof)
- 1X PBS
Procedures
- Prepare several cleaned tip-box caps by following wash: 100% EtOH rinse + di-H2O rinse, repeat once.
- Fill up the wash reagents into the caps. Label the box accordingly.
- Soak the slides in differnt reagents and gentle aggitation. Rinse the slide twice with diH2O in between. Time the soakig time and observe the change of slide.
Results
- The slide can not resist to either Acetone or Ethonal soaking. The array piece peared off by itself after 30 sec-40 sec of soaking.
- The slide can resist to 1N HCL soaking (> 1 min) without noticeable change of property.
Revised washing steps
- 1N HCL, 1 min -> 1st di-H2O, 1 min ->, 2nd di-H2O -> 1X PBS, 1 min -> dry up in hume hood, 5 - 10 min.
- The 1N KOH wasn't used neutrolize 1N HCL sicne the KOH residue may remian and interfere the downsteam applification such PCR reaction.
Microbead loading
Prodedures
- Start from the diluted and immobilized bead (with FITC labeled probes). Concentration: 1 mg/200 uL.
- The original bead info (Invitrogen): Dynabead M-280
- 10 mg (6-7 x 10^8 beads) -> 1 mg = 6-7 x 10^7 beads
- Resuspend by repeat pipetting using p200 pipettor.
- Dilute in 1/200 ratio with nuclease-free H2O
- Take 1 uL resuspension into a fresh 1.5 mL tube with 199 uL Nuclease-free H2O. Mix gently by repeat pipetting (p200).
- The new bead conc. will be 6 x 10^7 bead / 200 (uL) x 1/200 = 1500 bead /uL
- Leave the slide on Magnetic bord. Transfer 10 uL, 20 uL, 40 uL on microwell arrays.
- The bead drop can barely cover the entire array area except using 20 ~ 40 uL of total volume.
- Air day the slide on hume hood for until the diluent is fully evaporated (More than 10 min will be needed to dry up)
- It may be possible to dry on on magnetic hot plate at 60 degree
Microscope images
- The structure microwell and microbead were characterized using Zessis A1-mode flourescent microscope with 10X objective
FIG. 1: Black and white image (10X objective)
FIG. 2: Color image in green (10X objective)
Discussion
- No bead was found on microwell arrays in both B&W and color images. It is possible that:
- beads floated around on magnetic hot plate. The hot plate can't attract beads with enough magnetic force.
- The dilution of bead is not optimized ideally.
- Still some dust could be found on microwell arrays.
- Higher conc. of HCL or longer soaking time could be applied in more tests in the furture.