Daniel:Notebook/ComboLock/2016-3-16: Difference between revisions

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>Djacobse
>Djacobse
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#Wash 3X with Mineral Oil (no Span)
#Wash 3X with Mineral Oil (no Span)
##Remove oil phase with a pipette, and add in ~500 uL mineral oil
##Remove oil phase with a pipette, and add in ~500 uL mineral oil
##Pipette the oil and beads gently to mix; repeat
##Pipette the oil and beads gently to mix
##1.1 and 1.2
##Repeat 1.1 and 1.2
#
#Wash 3X with 6X SSC
##Remove supernatant and add 500 uL 6X SSC
##Pipette gently to mix
##Centrifuge for 1 min at 1000xg
##Repeat steps 2.1-2.3
#Add 600 uL of 2% Perfluoro-1-Octanol in 6X SSC (200 uL 20X SSC, 12 uL Perfluoro-1-Octanol, 388 uL ddH2O)
##Shake vigorously by hand for 20 seconds (seriously)
##Centrifuge for 1 min at 1000xg
##Remove supernatant and add an additional 500 uL 6X SSC
##Repeat 3.2 and 3.3 3 times

Revision as of 18:24, 16 March 2016

Drop Seq Test (Started 03/15/2016)

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After yesterday's results it would appear that we may have polyacrylamide droplets. However, I think the best test is a chemical one, so we'll be following the Macosko protocol for droplet breakage. This protocol uses Perfluoro-1-octanol to break apart droplets. If the droplets did form PA beads, then the octanol shouldn't have an effect. If they didn't the droplets should disappear.

Protocol

  1. Wash 3X with Mineral Oil (no Span)
    1. Remove oil phase with a pipette, and add in ~500 uL mineral oil
    2. Pipette the oil and beads gently to mix
    3. Repeat 1.1 and 1.2
  2. Wash 3X with 6X SSC
    1. Remove supernatant and add 500 uL 6X SSC
    2. Pipette gently to mix
    3. Centrifuge for 1 min at 1000xg
    4. Repeat steps 2.1-2.3
  3. Add 600 uL of 2% Perfluoro-1-Octanol in 6X SSC (200 uL 20X SSC, 12 uL Perfluoro-1-Octanol, 388 uL ddH2O)
    1. Shake vigorously by hand for 20 seconds (seriously)
    2. Centrifuge for 1 min at 1000xg
    3. Remove supernatant and add an additional 500 uL 6X SSC
    4. Repeat 3.2 and 3.3 3 times