Matt:LabNotes/2014-5-13

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MALAT1 Rolonies[edit]

  • Goal is to make more artificial MALAT1 Rolonies for padlock probe hybridization testing
  • Test to see the effect of increasing padlock probe concentration (10pM -> 100pM)

Procedure[edit]

  1. After fixing with 10% formalin 15min 37C & then sitting in 70% EtOH, Wash twice with 1X PBS
  2. Add 140ul 100nM MALAT1_template to wells C2 & B4
    • Pre-heat to 85C
    • Incubate @RT for 15 min
  3. Aspirate & Wash once with 1X PBS
  4. Capture with 100pM ppMALAT1_dcProbe1
    • Incubate @45C for 24 hours
Component Volume
10X Ampligase Buffer 20
1nM ppMALAT1_dcProbe1 20
5 U/ul Ampligase 20
H2O 140
Total 200
  1. Aspirate NO WASH
  2. Add RCA primer: 1ul FISSEQ_ppRCA in 199ul 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 171
10X Phi29 Buffer 20
25mM dNTP 2
2mM aa-dUTP 4
Phi29 3
Total 200
  1. Wash with 1X PBS once
  2. 4ul BS(PEG)9 in 196ul 1X PBS
    • Incubate 1hr @ RT
  3. Wash with 1X PBS twice
  4. Quench with 1M Tris pH 8.0 @RT for 30min
  5. Wash with 1X PBS twice

Detection[edit]

  1. Add 0.5uM dcProbe1-Cy3 in 30% formamide + 2X SSC
    • Preheat to 75C for 5min
    • Incubate @RT for 10min
  2. Wash with 2X SSC twice
  3. Image with Confocal (Saved in 5-16-2014)
    • Laser 3% & Gain 550
    • Pinhole = 1.0
  4. Strip with 80% formamide for 10min @RT
    • Preheated to 75C
  5. Wash with H2O twice

Results[edit]

Position Rolonies
1 472
2 566
3 334
4 3221
5 2816
6 2813
  • Comparing 3% vs 5% Laser
    • Position1 Laser 5%: 905 rolonies
    • Position1 Laser 3%: 472 rolonies

Conclusion[edit]

  • Compared to using 10pM ppMALAT1
    • Expect to see a ~10X increase in MALAT1 rolonies (from 100 to 1,000)
    • Definitely increased in number of rolonies but wasn't consistent
  • Although conditions were exactly the same, 10 times more rolonies in C2 well than B4
    • Guessing this is because cells in C2 were denser (more confluent)

Example BF Image of B4 vs C2

B4 C2
Position 2 Position 5
File:Pos2 B4 MALAT1Rol dcProbe1 L3 G550 Cy3 z01 ch01.jpg File:Pos5 C2 MALAT1Rol dcProbe1 L3 G550 Cy3 z01 ch01.jpg

Cell density very important in number of artificial rolonies that can be made