Sam:LabNotes/Microbione/2009-2-12: Difference between revisions
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==Objective== | ==Objective== | ||
*Clean and sterilize the slides before beads loading. | *Clean and sterilize the slides before beads loading. | ||
*Load the bead on slides and | *Load the bead on slides and evaluating the slide quality, bead arrangement under microscope. | ||
Line 26: | Line 26: | ||
*Fill up the wash reagents into the caps. Label the box accordingly. | *Fill up the wash reagents into the caps. Label the box accordingly. | ||
*Soak the slides in | *Soak the slides in different reagents and gentle agitation. Rinse the slide twice with diH2O in between. Time the soaking time and observe the change of slide. | ||
==Results== | ==Results== | ||
*The slide can not resist to either Acetone or | *The slide can not resist to either Acetone or Ethanol soaking. The array piece peeled 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. | *The slide can resist to 1N HCL soaking (> 1 min) without noticeable change of property. | ||
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==Revised washing steps== | ==Revised washing steps== | ||
* 1N HCL, 1 min -> 1st di-H2O, 1 min ->, 2nd di-H2O -> 1X PBS, 1 min -> dry up in | * 1N HCL, 1 min -> 1st di-H2O, 1 min ->, 2nd di-H2O -> 1X PBS, 1 min -> dry up in fume hood, 5 - 10 min. | ||
**The 1N KOH wasn't used | **The 1N KOH wasn't used to neutralize 1N HCL since the KOH residue may remain and interfere the downstream application such PCR reaction. | ||
Line 46: | Line 46: | ||
== | ==Procedures== | ||
*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. | ||
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*Leave the slide on Magnetic | *Leave the slide on Magnetic board. 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. | ** The bead drop can barely cover the entire array area except using 20 ~ 40 uL of total volume. | ||
*Air day the | *Air day the slides in fume hood until all of the diluent are 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== | ||
*The structure microwell and microbead were characterized using Zessis A1-mode | *The structure microwell and microbead were characterized using Zessis A1-mode fluorescent microscope with 10X objective | ||
Latest revision as of 17:26, 15 February 2009
Test of Microwell Slide Washing and Microbeads Loading[edit]
Objective[edit]
- Clean and sterilize the slides before beads loading.
- Load the bead on slides and evaluating the slide quality, bead arrangement under microscope.
Slide washing[edit]
Reagents[edit]
- 1N HCL (diluted from 12N to 1N)
- Acetone
- Ethanol (200-proof)
- 1X PBS
Procedures[edit]
- 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 different reagents and gentle agitation. Rinse the slide twice with diH2O in between. Time the soaking time and observe the change of slide.
Results[edit]
- The slide can not resist to either Acetone or Ethanol soaking. The array piece peeled 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[edit]
- 1N HCL, 1 min -> 1st di-H2O, 1 min ->, 2nd di-H2O -> 1X PBS, 1 min -> dry up in fume hood, 5 - 10 min.
- The 1N KOH wasn't used to neutralize 1N HCL since the KOH residue may remain and interfere the downstream application such PCR reaction.
Microbead loading[edit]
Procedures[edit]
- Start from the diluted and immobilized bead (with FITC labeled probes). Concentration: 1 mg/200 uL.
- The original bead info (Invitrogen): Dynabead M-280; Dynabead application
- 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 board. 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 slides in fume hood until all of the diluent are 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[edit]
- The structure microwell and microbead were characterized using Zessis A1-mode fluorescent microscope with 10X objective
FIG. 1: Black and white image (10X objective)
FIG. 2: Color image in green (10X objective)
Discussion[edit]
- 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.