Hosuk:LabNotesMDA/2013-6-3: Difference between revisions

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*Reduce mixing speed, with lower ALS concentration and longer denature time
*Reduce mixing speed, with lower ALS concentration and longer denature time
*Jeff’s suggestion : 133mM of ALS after mixing with cell.
*Jeff’s suggestion : 133mM of ALS after mixing with cell.
*Mixer volume/ (Mixer volume – cell capture volume) = 1.12 --> Required concentration of KOH for 133mM = '''148.96 mM''' (~ 150mM)
*Mixer volume/ (Mixer volume – cell capture volume) = 1.12 --> Required concentration of KOH in prepaired ALS tube for 133mM = '''148.96 mM''' (~ 150mM)
 


=====To reduce sample loss=====
=====To reduce sample loss=====

Revision as of 16:59, 3 June 2013

Actions for next run

To get larger fragment size
  • This is the first priority
  • Reduce mixing speed, with lower ALS concentration and longer denature time
  • Jeff’s suggestion : 133mM of ALS after mixing with cell.
  • Mixer volume/ (Mixer volume – cell capture volume) = 1.12 --> Required concentration of KOH in prepaired ALS tube for 133mM = 148.96 mM (~ 150mM)
To reduce sample loss
  • Push the volume in ring mixer with air, and use MDA master mix filled in mixer and run MDA
  • For the next device design, add inlet between mixer and compartment for extracting MDA product from ring mixer
  • Total Compartments volume will be almost matched with the volume of ring mixer, as usual.


To minimize bias
  • Adjust (decrease) concentration of phi29 and primers.


Changes for next device design
  • Add inlet channels between mixer and compartment, and one more inlet next to ALS for air input
  • Slightly increase mixer volume --> will check how much volume will be transferred from current ring mixer to compartments using actual ALS with 10min. mixing