Matt:LabNotes/2014-9-8

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Revision as of 21:11, 10 September 2014 by >Mzcai
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in situ Padlock Probe Capture Temp: 45C vs 60C

Procedure

  • PGP1F Samples with 1st Rolonies prepared by Hosuk
    • Fixed 9/6/14 and 1st Rolonies on 9/8/14
  • Add 100ul Padlock Probe Mix
Components Volume
5 U/ul Ampligase 10
10X Ampligase Buffer 10
100uM ppCOL1A1 2
H2O 78
Total 100
  • Incubate at 37C for 30min
  • Incubate at 45C or 60C for 26hrs
  • Wash with 1X PBS twice
  • Add 200ul 0.5uM FISSEQ_ppRCA in 2X SSC + 30% formamide (pre-heated to 75C) and incubate at 45C for 30min
  • Wash with 2X SSC twice
  • Add 200ul RCA Mix and incubate at 30C for 16hrs
    • H2O : 172uL
    • 10x Buffer : 20uL
    • dNTP : 2uL
    • a-dUTP : 4uL
    • Phi29 (100 U/µl) : 2uL
  • Wash with 1X PBS once
  • Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) at RT for 1hr
  • Wash with 1X PBS twice
  • Add 200ul 1M Tris pH 8.0 at RT for 30min
  • Wash with 1X PBS twice and store in PBS