Matt:LabNotes/2014-4-30

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Revision as of 20:07, 7 May 2014 by >Mzcai (Created page with "==MALAT1 Rolonies and EDTA== ===Procedure=== #Add 140ul 100nM MALAT1_template to each well #*Pre-heat to 85C #*Incubate @RT for 15 min #Aspirate & Wash once with 1X PBS #Capt...")
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MALAT1 Rolonies and EDTA

Procedure

  1. Add 140ul 100nM MALAT1_template to each well
    • Pre-heat to 85C
    • Incubate @RT for 15 min
  2. Aspirate & Wash once with 1X PBS
  3. Capture with 10pM ppMALAT1
    • Incubate @45C for 20 hours
Component Volume
10X Ampligase Buffer 60
1nM ppMALAT1 6
5 U/ul Ampligase 60
H2O 474
Total 600
  1. Aspirate NO WASH
  2. Add RCA primer: 3ul FISSEQ_ppRCA in 597 2X SSC/30%
    • Pre-heat to 60C
    • Add 100ul @45C for 15min
    • Wash 2X SSC twice
    • Wash 0.1X SSC twice
  3. Add RCA mix
    • Incubate 22hrs @ 30C
Component Volume
H2O 513
10X Phi29 Buffer 60
25mM dNTP 6
2mM aa-dUTP 12
Phi29 9
Total 600
  1. Wash 1X PBS once
  2. 12ul BS(PEG)9 in 588ul 1X PBS
    • Incubate @RT 1hr
  3. Wash 1X PBS twice
  4. Quench with 1M Tris pH 8.0 @RT 1hr
    • To A3-A5: 1M Tris pH 8.0
    • To D3-D5: 0.9M Tris + 50mM EDTA ph 8.0 (45mL 1M Tris + 5mL 500mM EDTA)
  5. Wash 1X PBS twice
  6. Add 1uM dcProbe2-Cy3 in 30% formamide + 2X SSC
    • Preheat to 75C for 5min
    • Incubate @RT for 10min
  7. Wash 2X SSC twice
  8. Image
  9. Strip w/ 80% formamide in 2X SSC
    • Pre-heat to 75C
    • Incubate @RT for 15min
  10. Wash with ddH2O twice

Results