Hosuk:LabNotesMDA/2013-6-28: Difference between revisions

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*In standard 20uL reaction, ALS is used 1.5uL for 1uL of cell, so 1.5/2.5 x 400mM KOH = 240mM in ALS+Cell while denaturing.
*In standard 20uL reaction, ALS is used 1.5uL for 1uL of cell, so 1.5/2.5 x 400mM KOH = 240mM in ALS+Cell while denaturing.
*Therefore current 400mM of KOH in ALS is much higher concentration for MDA device, because KOH conc. in ring mixer while denaturing is 357mM.
*Therefore current 400mM of KOH in ALS is much higher concentration for MDA device, because KOH conc. in ring mixer while denaturing is 357mM.
**Final KOH conc. in ring mixer =(KOH conc. in ALS buffer)/1.12  (for current device ver. 7.3)
*Let’s try 240mM in ring mixer!
*Let’s try 240mM in ring mixer!
[[File:ALS_NS_recipe.png|500px]]

Latest revision as of 18:16, 28 June 2013


MDA with PGP1F, 6th[edit]

  • MDA run for 8hr @06/27
  • 400mM ALS --> original protocol
  • Slow mixing --> 1 sec period, mixing for 5min
  • Run MDA in ring mixer --> 25th chamber!
  • Push ring mixer volume with air
    • Left 8 chamber were empty, too many empty chamber...


Result[edit]

  • qPCR result looked good.
  • Most of chambers have 500pg~2400pg of amplicons.
  • Left 8 chamber group were nearly empty, so that's why ch6 has almost no amplicon was detected.
  • 25th chamber(Ring mixer) had less amount than other chamber did, but there was amplicon!
  • It seemed that low conc. of ALS doesn’t work in the MDA device.


File:QPCR Result 2013-06-28 Edited.png File:QPCR Result MassTable 2013-06-28.png


CoRE fragmentation[edit]

  • Will be done @06/28


Considerations[edit]

  • In standard 20uL reaction, ALS is used 1.5uL for 1uL of cell, so 1.5/2.5 x 400mM KOH = 240mM in ALS+Cell while denaturing.
  • Therefore current 400mM of KOH in ALS is much higher concentration for MDA device, because KOH conc. in ring mixer while denaturing is 357mM.
    • Final KOH conc. in ring mixer =(KOH conc. in ALS buffer)/1.12 (for current device ver. 7.3)
  • Let’s try 240mM in ring mixer!


File:ALS NS recipe.png