Matt:LabNotes/2014-9-6

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Revision as of 11:24, 16 September 2014 by >Mzcai
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"Tertiary" Rolony Synthesis

  • Hypothesis: We can increase the number of gene-specific rolonies (previously known as secondary rolonies) by repeating padlock probe capture and rolonization on samples with primary and secondary rolonies
    • There should be more targets for the padlock probes to capture since all primary rolonies are still fixed and 100% of secondary rolonies will be possible targets for padlock probes
    • May increase amplification bias

Procedure

  • Add 100ul Padlock Probe Mix (14pM per padlock probe)
Components Volume
5 U/ul Ampligase 10
10X Ampligase Buffer 10
908 nM Agi26k0gap 18.6
H2O 61.4
Total 100
  • Incubate at 37C for 30min
  • Incubate at 45C for 26hrs
  • Wash with 1X PBS twice
  • Add 200ul 0.5 uM FISSEQ_ppRCA primer in 2X SSC (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

7 Cycle 3-Dye Decoding

  • Saved in 9-9-2014