Hosuk:LabNotesMDA/2013-6-28
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MDA with PGP1F, 6th
- 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
- 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
- Will be done @06/28
Considerations
- In 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.
- Let’s try 240mM in ring mixer!