Matt:LabNotes/2014-5-7

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ppMALAT1_dcProbe1 Capture of MALAT1 Rolonies +/- EDTA[edit]

  • Continued from Matt:LabNotes/2014-4-30
  • Test to see if rolonies quenched with TE (Tris + EDTA) are easier for padlock probes to hybridize to
  • Quantify the amplification of Secondary Rolonization from Primary Rolonies

Procedure[edit]

  • Add 100ul ppMALAT1_dcProbe1 with 0.5 U/ul Ampligase
    • A3&D3: 10pM
    • A4&D4: 50pM
    • A5&D5: 2uM
    • Incubate @45C for 24 hours

10pM

Component Volume
10X Ampligase Buffer 20
5 U/ul Ampligase 20
1nM ppMALAT1_dcProbe1 2
H2O 158
Total 200

50pM

Component Volume
10X Ampligase Buffer 20
5 U/ul Ampligase 20
1nM ppMALAT1_dcProbe1 10
H2O 150
Total 200

2uM

Component Volume
10X Ampligase Buffer 20
5 U/ul Ampligase 20
100uM ppMALAT1_dcProbe1 4
H2O 156
Total 200
  • Aspirate NO WASH
  • 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
  • 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
  • Wash 1X PBS once
  • 12ul BS(PEG)9 in 588ul 1X PBS
    • Incubate @RT 1hr
  • Wash 1X PBS twice
  • Quench with 1M Tris pH 8.0 @RT 1hr
  • Wash 1X PBS twice
  • Add 1uM dcProbe1-Cy3 in 30% formamide + 2X SSC
    • Preheat to 75C for 5min
    • Incubate @RT for 10min
  • Wash 2X SSC twice
  • Image
    • Used Pinhole: 2.0

Results[edit]

  • Saved in 5-8-2014
  • Used Pinhole = 2.0
    • Tried Pinhole = 1.0 for Pos9 the count of rolonies was 2,801 (80% of Pinhole 2.0)
Well Position Rolonies
A3 1 930
A3 2 1562
A4 3 3134
A4 4 4352
A5 5 4997
A5 6 9189
D3 7 1272
D3 8 1062
D4 9 3480
D4 10 3758
D5 11 3315
D5 12 1909

File:MALAT1 2ndRol Results.JPG

Conclusion[edit]

  • Padlock probe capture (followed by RCA) of primary rolonies greatly amplifies signal
  • EDTA does not help with padlock probe capture
  • Increasing concentration above 50pM does not improve 2nd rolony yield
  • Based on Matt:LabNotes/2014-4-30#Results there's good correlation with number of primary rolonies and number of secondary rolonies