Matt:LabNotes/2014-10-6

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Testing Agi26k0gap + suppressor Oligos & Agi26k20gap[edit]

Sample[edit]

  • Agi26k0gap Control: PGP1F Fix 5.19.14 S2
  • Agi26k0gap + suppressors: PGP1F Fix 5.19.14 S3
  • Agi26k20gap: Fix 10.1.14

Procedure[edit]

  • Agi26k0gap Control
Components Volume
5 U/ul Ampligase 10
10X Ampligase Buffer 10
Agi26k0gap 35
H2O 45
Total 100
  • Agi26k0gap + suppressors
Components Volume
5 U/ul Ampligase 10
10X Ampligase Buffer 10
Agi26k0gap 35
600 nM (100nM each) supp_Mix 5
H2O 40
Total 100
  • Agi26k20gap
Components Volume
5 U/ul Ampligase 10
10X Ampligase Buffer 10
Agi26k20gap 70
25mM dNTP 1
10 U/ul KlenTaq 2
H2O 7
Total 100
  • Incubate at 37C for 30 min
  • Incubate at 60C for 24 hrs
  • 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 : 173uL
    • 10x Buffer : 20uL
    • dNTP : 2uL
    • a-dUTP : 4uL
    • Phi29 (1000 U/µl) : 1uL
  • 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

Decoding Results[edit]