Hosuk:LabNotes/2015-1-20

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Test - New Padlock Probes[edit]

  • Test new padlock probes – hybridize to cDNA and ampligase, then RCA one time
  • Matt produced CA12k_Nov2014_V4_Contig and CA12k_Nov2014_V7_Const
    • V4 concentration is 4 times higher than V7


  • I prepared 4 PGP1F P17 on MatTek dishes (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 5 min, then 0.1N HCL for 2 min.
  • Do RT (without a-dUTP) – start at 11am 01/19 to 11pm 01/19
  • Run RNase H : 10:30am 01/20
  • Run Ampligase 11:30am 01/20
    • S1 : V7 --> at 37C for 30min, then 60C for ~4hr
    • S2 : V4 --> at 37C for 30min, then 60C for ~4hr
    • S3 : Agilent --> No agilent padlock probes left, so I use H2O instead
    • S4 : 1st Rolony generation
      • Run RCA for S4 --> 1pm 01/20
      • Finish with BS(PEG)9 and Tris pH 8.0 --> 11:00am 01/21
  • Run RCA for S1, S2, S3 --> 11:40am 01/21