Sam:LabNotes/Microbione/2009-2-12
Jump to navigation
Jump to search
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.