Hosuk:LabNotesMDA/2013-6-3: Difference between revisions
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>Hosuki78 No edit summary |
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*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 in prepaired ALS tube for 133mM = '''148.96 mM''' (~ 149mM) | *Mixer volume/ (Mixer volume – cell capture volume) = 1.12 --> Required concentration of KOH in prepaired ALS tube for 133mM = '''148.96 mM''' (~ 149mM) | ||
{| {{table}} | |||
| | |||
|| | |||
| 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 | |||
|} | |||
{| {{table}} | |||
| | |||
|| | |||
| 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===== | =====To reduce sample loss===== |
Revision as of 17:15, 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 (~ 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