Hosuk:LabNotesMDA/2013-6-28: Difference between revisions
Jump to navigation
Jump to search
>Hosuki78 |
>Hosuki78 |
||
(One intermediate revision by the same user not shown) | |||
Line 32: | Line 32: | ||
*In standard 20uL reaction, ALS is used 1.5uL for 1uL of cell, so 1.5/2.5 x 400mM KOH = 240mM in ALS+Cell while denaturing. | *In standard 20uL reaction, ALS is used 1.5uL for 1uL of cell, so 1.5/2.5 x 400mM KOH = 240mM in ALS+Cell while denaturing. | ||
*Therefore current 400mM of KOH in ALS is much higher concentration for MDA device, because KOH conc. in ring mixer while denaturing is 357mM. | *Therefore current 400mM of KOH in ALS is much higher concentration for MDA device, because KOH conc. in ring mixer while denaturing is 357mM. | ||
**Final KOH conc. in ring mixer =(KOH conc. in ALS buffer)/1.12 | **Final KOH conc. in ring mixer =(KOH conc. in ALS buffer)/1.12 (for current device ver. 7.3) | ||
*Let’s try 240mM in ring mixer! | *Let’s try 240mM in ring mixer! | ||
[[File:ALS_NS_recipe.png|500px]] | [[File:ALS_NS_recipe.png|500px]] |
Latest revision as of 18:16, 28 June 2013
MDA with PGP1F, 6th[edit]
- MDA run for 8hr @06/27
- 400mM ALS --> original protocol
- Slow mixing --> 1 sec period, mixing for 5min
- Run MDA in ring mixer --> 25th chamber!
- Push ring mixer volume with air
- Left 8 chamber were empty, too many empty chamber...
Result[edit]
- qPCR result looked good.
- Most of chambers have 500pg~2400pg of amplicons.
- Left 8 chamber group were nearly empty, so that's why ch6 has almost no amplicon was detected.
- 25th chamber(Ring mixer) had less amount than other chamber did, but there was amplicon!
- It seemed that low conc. of ALS doesn’t work in the MDA device.
File:QPCR Result 2013-06-28 Edited.png File:QPCR Result MassTable 2013-06-28.png
CoRE fragmentation[edit]
- Will be done @06/28
Considerations[edit]
- In standard 20uL reaction, ALS is used 1.5uL for 1uL of cell, so 1.5/2.5 x 400mM KOH = 240mM in ALS+Cell while denaturing.
- Therefore current 400mM of KOH in ALS is much higher concentration for MDA device, because KOH conc. in ring mixer while denaturing is 357mM.
- Final KOH conc. in ring mixer =(KOH conc. in ALS buffer)/1.12 (for current device ver. 7.3)
- Let’s try 240mM in ring mixer!