Hosuk:LabNotes/2014-4-14

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PGP1F sample for 13k Padlock probe test (--> Agi26k0gap)[edit]

Ampligase[edit]

  • Matt’s Agi26k0gap Padlock Probe set
    • Padlock probes (16.5pmole) : 97uL (--> 170pM total --> 13 fM per each)
    • Ampligase Buffer : 15uL
    • Ampligase Enzyme : 15uL
    • H2O : 23uL
    • Total 150 uL
  • Start Ampligase: 04/11/14, 10:30am, incubate for 48 hr.


2nd RCA reaction[edit]

  • RCA Mix
    • H2O : 162uL
    • 10x Buffer : 20uL
    • dNTP : 4uL
    • adUTP : 8u:
    • Phi29 : 6uL
  • Start RCA : 04/13/14 ~ 04/14/14 for 24hr.


Imaging & Decoding[edit]

  • Imaging : 04/14/14
  • 1uM of FAM, and Cy3 in 100uL total dcProbes volume
    • 10uL of FAM + 10uL of Cy3 + 80uL of 2x SSC


  • 20x obj. 2048 x 2048 resolution
  • Laser : 5% for both 488, 532nm
  • PMT gain : 610


  • Step0 : Cy3-1st Rololy detection
  • Step1 : dcProbe0-FAM, dcProbe0-Cy3
  • Step1 : dcProbe1-FAM, dcProbe1-Cy3

...

  • Step5 : dcProbe4-FAM, dcProbe4-Cy3


  • Strip with 80% formamide at inbetween each step (for 15min incubation)


Decoding Result[edit]

  • Each image at each step showed reasonable number of signals, but not many.
  • 1st Rolony signal was too much decreased compared to the first observation at 04/10... WHY?
  • Analysis is on going
  • Temp. result(04/14) : DecodingResults_v2.xlsx