Matt:LabNotes/2014-4-30

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MALAT1 Rolonies and EDTA[edit]

  • Test to see if rolonies quenched with TE (Tris + EDTA) are easier for padlock probes to hybridize to
    • Use artificial MALAT1 rolonies
  • Quantify the amplification of Secondary Rolonization from Primary Rolonies

Procedure[edit]

  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[edit]

  • PISA wasn't able to count many images due to not reaching minimum number of features
    • Used bwlabel to count features after applying a threshold of 9,000 to make a bw image
Well Position Rolonies counted by PISA Rolonies counted by bwlabel (threshold 9000)
A3 1 N/A 55
A3 2 140 141
A4 3 N/A 69
A4 4 N/A 104
A5 5 N/A 66
A5 6 N/A 127
D3 7 N/A 41
D3 8 N/A 51
D4 9 N/A 48
D4 10 137 146
D5 11 139 140
D5 12 128 127