Hosuk:LabNotesMDA/2013-2-5: Difference between revisions
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**2/4 samples: 5 tubes by taking 0.5uL from four tubes and merging into one sample | **2/4 samples: 5 tubes by taking 0.5uL from four tubes and merging into one sample | ||
*PCR master mix | *PCR master mix | ||
**KAPA fast for 30uL reaction | **KAPA fast for 30uL reaction --> this was not used! | ||
***KAPA fast 15 uL | ***KAPA fast 15 uL | ||
***10 uM primer 1 .6 uL | ***10 uM primer 1 .6 uL |
Revision as of 17:40, 6 February 2013
qPCR run
- Run qPCR with the sample from 1/28, 1/29 and 2/4 using mouse and human primer
- The number of tubes
- 1/28: 16ea (#9~#24)
- 1/29: 21ea (#1~#21)
- 2/4: 23ea (#2~#24)
- Preparation
- 1/28, 1/29 samples: 8 tubes by taking 0.5uL from two tubes and merging into one sample
- 2/4 samples: 5 tubes by taking 0.5uL from four tubes and merging into one sample
- PCR master mix
- KAPA fast for 30uL reaction --> this was not used!
- KAPA fast 15 uL
- 10 uM primer 1 .6 uL
- 10 uM primer 2 .6 uL
- DNA from library 2 uL
- water 11.4 uL
- total 30 uL
- For 20uL reaction with mouse primer
- KAPA fast 10 uL
- 10 uM primer 1 .4 uL
- 10 uM primer 2 .4 uL
- DNA from library 1 uL
- water 8.2 uL
- total 20 uL
- For 20uL reaction with mouse primer
- KAPA fast 10 uL
- 10 uM primer 1 .4 uL
- 10 uM primer 2 .4 uL
- DNA from library 2 uL
- water 7.2 uL
- total 20 uL
- KAPA fast for 30uL reaction --> this was not used!
PCR reaction
- 95C 10sec
- 58C 20sec
- 72C 45sec
- 40cycle
Result
- Most of curves rose after 24 cycle, and became flat around 32 cycle
- Seemed too much low amount of MDA product
- Human DNA has also been seen.
- (need more description)