Sam:LabNotes/Microbiome-new/2009-3-12: Difference between revisions

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>Sam Chiang
(New page: ='''Test of Microwell Slide Washing and Microbeads Loading'''= ==Objective== *Clean and sterilize the slides before beads loading. *Load the bead on slides and evaluating the slide qual...)
 
>Sam Chiang
(Removing all content from page)
 
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='''Test of Microwell Slide Washing and Microbeads Loading'''=


==Objective==
*Clean and sterilize the slides before beads loading.
*Load the bead on slides and evaluating the slide quality, bead arrangement under microscope.
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='''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 different reagents and gentle agitation. Rinse the slide twice with diH2O in between. Time the soaking time and observe the change of slide.
==Results==
*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==
* 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.
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='''Microbead loading'''=
==Procedures==
*Start from the diluted and immobilized bead (with FITC labeled probes). Concentration: 1 mg/200 uL.
** The original bead info (Invitrogen): [[Media:Dynabead M-280.pdf|Dynabead M-280]]; [[Media:Dynabead application.pdf|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==
*The structure microwell and microbead were characterized using Zessis A1-mode fluorescent microscope with 10X objective
'''FIG. 1: Black and white image (10X objective)'''
[[Image:blackwhite 2-13-09_bmp.bmp|600px|none]]
'''FIG. 2: Color image in green (10X objective)'''
[[Image:color 2-13-09_bmp.bmp|600px|none]]
==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.

Latest revision as of 08:54, 21 March 2009