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