Hosuk:LabNotesMDA/2013-6-3
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Actions for next run[edit]
To get larger fragment size[edit]
- 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[edit]
- 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[edit]
- Adjust (decrease) concentration of phi29 and primers.
Changes for next device design[edit]
- 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
Test new ALS and longer Mixing, and volume transfer by air[edit]
Cell lysis test with new ALS recipe[edit]
- HeLa cell was used prior to using PGP1.
- 1 sec valving period --> 5 times slower than usual
- Cell was not lysed immediately, but after 1 cycle in ring mixer, cell began to be lysed
Volume transfer from Mixer to Compartment by pushing Air after 10min. mixing[edit]
- About 3 compartment volumes were not filled.
- ALS+Cell solution was remained on the wall of ring mixer.
- For the next real run, just filling compartment as much as possible, and run rest of procedure.
- First check point is getting bigger fragment by reducing sheer stress.
- And another option is
- Close valves in between compartment, then fill water in ring mixer
- Then fill the empty compartment, so that remaining of mixer could be flushed and go to empty compartments.
MDA Run with PGP1F (3rd)[edit]
- Same procedure with Eric's LabNote @ 05/20, EXCEPT
- ALS mixing time --> 10min. with 1 sec valving period (previously 200ms)
- Push the ALS+cell in mixer to compartment with air (start from 1.8psi, and 2.5psi at the end)
- Because of remaining in mixer, first (left) 6 compartment were empty (I filled solution at the last compartment, so chamber 1~6 became empty)
- Run MDA for 10hr.