Hosuk:LabNotesMDA/2013-6-3

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Revision as of 17:15, 3 June 2013 by >Hosuki78
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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 (~ 149mM)


ALS, current ALS, new
H2O 69.5 uL 74.52 uL
1M DTT 10 uL 10 uL
5M KOH 8 uL 2.98 uL
8% Tween20 12.5 uL 12.5 uL


NS, current NS, new
H2O 0 uL 54.9 uL
NS stock 87.5 uL 32.6 uL
8% Tween20 12.5 uL 12.5 uL


    • NS stock : 4mL of 1M HCl + 6mL of 1M TrisHCl


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