Hosuk:LabNotes/2015-1-18

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Test new BS(PEG)9 and a-dUTP[edit]

Need to test[edit]

  • I’ve ordered a new a-dUTP (Anaspec) and BS(PEG)9, but they were sitting in a room temp. for 2~3 days exposed to sun light. So I want to test the BS(PEG)9 and a-dUTP are fine.


Proceduer[edit]

  • I prepared 2 PGP1F P17 on MatTek dish (diameter = 10mm)
  • Take out the dishes from CERC to the lab, and fix them with 10% Formaldehyde for 15min at 37C.
  • Wash with PBS 3 times, then incubate with 70% EtOH for 2 min, then 0.1N HCL for 2 min.
  • Do RT (without a-dUTP) – start at 10am 01/16 to 8pm 01/16
  • Run RCA at 12am 01/17
    • S1 : new BS(PEG)9 and new a-dUTP
    • S2 : old BS(PEG)9 and old a-dUTP
  • Finish all steps – 5:30pm 01/17
  • Image – 11am 01/18
    • 20x : Pinhole = 2, Laser = 4, PMT = 540
    • 63x : Pinhole = 1, Laser = 4, PMT = 540


Result[edit]

  • S2(old reagent) seems better than S1(new reagent), but not much difference between old and new set! – new a-dUTP (Anaspec) and BS(PEG)9 can be used!
  • However, all signal looks weaker than normal look of 1st rolony results that I’ve got so far, especially signal excited by Laser power = 4 should be seen much stronger than this.
  • I’ll test a new Padlock Probes.