Sam:LabNotes/Microbione/2009-2-12: Difference between revisions

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>Sam Chiang
>Sam Chiang
 
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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 evaluting the slide quality, bead arrangement under microscope.
*Load the bead on slides and evaluating the slide quality, bead arrangement under microscope.
 
 
----
 
='''Slide washing'''=
 




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*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 differnt reagents and gentle aggitation. Rinse the slide twice with diH2O in between. Time the soakig time and observe the change of slide.
*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 Ethonal soaking. The array piece peared off by itself after 30 sec-40 sec of soaking.
*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 hume hood, 5 - 10 min.
* 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.
**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'''=


=Microbead loading=




 
==Procedures==
==Prodedures==


*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]]; [[Media:Dynabead application.pdf|Dynabead application]]
** 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


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*Leave the slide on Magnetic bord. Transfer 10 uL, 20 uL, 40 uL on microwell arrays.  
*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 slide on hume hood for until the diluent is fully evaporated


*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==


==Microscope image==
*The structure microwell and microbead were characterized using Zessis A1-mode fluorescent microscope with 10X objective


*The structure microwell and microbead were characterized using Zessis A1-mode flourescent microscope with 10X objective


'''FIG. 1: 10X Black and white image'''
'''FIG. 1: Black and white image (10X objective)'''
[[Image:blackwhite 2-13-09_bmp.bmp|600px|none]]
[[Image:blackwhite 2-13-09_bmp.bmp|600px|none]]


'''FIG. 2: 10X Color image in green'''
 
[[Image:color 2-13-09.tif|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 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.


  • 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:
  1. beads floated around on magnetic hot plate. The hot plate can't attract beads with enough magnetic force.
  2. The dilution of bead is not optimized ideally.


  • Still some dust could be found on microwell arrays.
  1. Higher conc. of HCL or longer soaking time could be applied in more tests in the furture.