Hosuk:LabNotesMDA/2013-6-28: Difference between revisions
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==== | ====Result==== | ||
* | *qPCR result looked good. | ||
*Most of chambers have 500pg~2400pg of amplicons. | *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. | *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! | *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|800px]] [[File:qPCR_Result_MassTable_2013-06-28.png|250px]] | [[File:qPCR_Result_2013-06-28_Edited.png|800px]] [[File:qPCR_Result_MassTable_2013-06-28.png|250px]] | ||
====CoRE fragmentation==== | ====CoRE fragmentation==== | ||
* | *Will be done @06/28 | ||
====Considerations==== | |||
*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! | |||
[[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!