Hosuk:LabNotes/2013-11-18

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Experimental Plan[edit]

Rolony in MatTek dish, 96 well plate[edit]

  • MatTek dish
    • Fix :
    • Aspirate media, Add 10% Formalin, Incubate 37C for 15min.
    • Wash with PBS 3 time, Add 2mL Ethanol


  • 96 well plate
    • In T25 flask, Add 1mL Trypsin, Incubate 37C for 5min.
    • Add 4mL media (Total 5mL), Take 50uL, add in each well
    • Fix by aspirating media, adding 10% Formalin


  • Check cells after Fixation


Dye labeled Probe Annealing Test[edit]

  • Check Non-specific binding, and Upper limit
    • Use dcProbe#-Cy3 --> check non-specific binding
    • Use Cy5-1st Rolony --> Upper limit
  • Dye-Probe mixed with different concentration of Formamide
    • 10%, 30%, 50%, 70% in 2x SSC
  • Incubating temperature
    • ??


  • Check the number of Rolonies


Padlock Probe Concentration Test[edit]

  • Lower limit, Upper limit of the concentration
  • Normally 0.2uM for each Padlock Probe after PCR
  • Use One MatTek Dish
  • Use ppMALAT1
    • 0.1uM, 5uM, and check the signal, then test middle concentration
  • Preheat ppMALAT1 at 80C
  • Incubate at 50C for 15min


Ampligase test[edit]

  • Use one MatTek dish
  • Add Padlock probes, Ligase, 2nd RCA and check how many 2nd Rolonies


  • Ampligase reaction condition
    • Add 3 Padlock probes – ppACTB, ppRAB7A, ppMALAT1
    • Padlock probe concentration : 1uM each (? could be changed)
    • Padlock probe incubation time : 30min
    • Ampligase concentration : 5U
    • Temperature : 60C
    • Incubation time : 4hr.


File:Plan 2013-11-18.png