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

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)

File:QPCR curve 2013-02-05.jpg