Matt:LabNotes/2014-3-19

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Padlock Probe Concentration Test using Fixed Template[edit]

  • 96-well plate NCI-H1975 Cells (C8, C9, C10, D8, D9, D10, E8, E9, E10, F9)
    • Alan already marked locations with good cell density
  • Test concentrations NegCtrl-0pM, 10pM, 50pM, 100pM, 10nM
    • 2 wells for each

Fixing Template in cells[edit]

  1. Pre-heat 10uM MALAT1_Template in 2X SSC to 85C (50ul 100uM MALAT1_Template + 50ul 20X SSC + 400ul H2O)
  2. Add 50ul to each well
    • Incubate @RT for 15min
  3. Aspirate but don't wash
  4. Add 50ul BS(PEG)9 mix (10ul BS(PEG)9 + 490ul 1X PBS)
    • Incubate @RT for 1hr
  5. Wash with 1X PBS twice
  6. Add 50ul 1M Tris pH 8.0
    • Incubate @ RT for 30min
  7. Wash with 1X PBS twice

Anneal Padlock Probes[edit]

  • 1X Ampligase Buffer
  • 0.5 U/ul Ampligase
  • Incubate @ 45C for 24 hours (Started at 4pm)
    • Wrapped in parafilm and put in ziploc with wet towel

Negative Control 0pM (C8 & C9)[edit]

Component Volume
10X Ampligase Buffer 10
5U/ul Ampligase 10
H2O 80
Total 100

10pM (D8 & D9)[edit]

Component Volume
10X Ampligase Buffer 10
1nM ppMALAT1 1
5U/ul Ampligase 10
H2O 79
Total 100

50pM (E8 & E9)[edit]

Component Volume
10X Ampligase Buffer 10
1nM ppMALAT1 5
5U/ul Ampligase 10
H2O 75
Total 100

100pM (C10 & D10)[edit]

Component Volume
10X Ampligase Buffer 10
1nM ppMALAT1 10
5U/ul Ampligase 10
H2O 70
Total 100

10nM (E10 & F9)[edit]

Component Volume
10X Ampligase Buffer 10
1uM ppMALAT1 1
5U/ul Ampligase 10
H2O 79
Total 100

Secondary RCA[edit]

  1. Aspirate but don't wash
  2. Pre-annealing the FISSEQ_ppRCA primer for the rolling circle amplification reaction.
    1. 2.5ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 497.5ul of 2xSSC/30% formamide
    2. Preheat primer at 60C (Tm = 55C when not in 30% formamide)
    3. Add 50ul and incubate at 45C for 15min.
    4. Aspirate, and wash using 2x SSC twice
    5. Aspirate, and wash using 0.1x SSC twice
  3. Prepare the rolling circle amplification reaction mixtures on ice.
    1. 427.5 ul ddH2O,
    2. 50 ul 10x Phi29 buffer,
    3. 5 ul 25 mM dNTP,
    4. 10 ul 2 mM aminoallyl dUTP
    5. 7.5 ul Phi29 DNA polymerase (Epicentre, low concentration)
      • 500ul Total Volume
  4. Add 50ul each well and incubate 20hrs at 30C. (Put in at 4:30pm 3/20/14)
  5. Wash 1X PBS once
  6. Add 50ul BS(PEG)9 mix to each well and incubate @RT for 1hr
  7. Wash 1X PBS twice
  8. Add 50ul 1M Tris pH 8.0 @RT for 30min
  9. Wash 1X PBS twice

Detection[edit]

  • Made 500ul of 1uM dcProbe2-Cy3 (495ul 2XSSC & 30%formmaide + 5ul 100uM dcProbe2-Cy3)
  • Pre-heated @75C for 5 min and then added 50ul to each well
  • Let sit @RT for 5 min
  • Washed twice with 2X SSC
  • Saved in 3-24-2014

Results[edit]

  • All imaging was done with 5% Laser and Gain of 550
    • C10_100pM had a Z-thickness of ~37 microns so tried taking both 20 and 30 z-slices
      • As seen in the table it didn't make a big difference
  • The disparity between C10 and D10 (both 100pM) can be partly attributed to different number of cells in field of view
C10 D10
File:C10 100pM G550 Cy3 z12 ch01.png File:D10 100pM G550 30slices Cy3 z09 ch01.png


Image Rolony Count
C8_NegCtrl_G550_Cy3_ch00_MIP 0
C9_NegCtrl_G550_Cy3_ch00_MIP 0
D8_10pM_G550_Cy3_ch00_MIP 51
D9_10pM_G550_Cy3_ch00_MIP 101
E8_50pM_G550_Cy3_ch00_MIP 355
E9_50pM_G550_Cy3_ch00_MIP 764
C10_100pM_G550_Cy3_ch00_MIP 3216
D10_100pM_G550_30slices_Cy3_ch00_MIP 521
D10_100pM_G550_Cy3_ch00_MIP 497
E10_10nM_G550_Cy3_ch00_MIP 17509
F9_10nM_G550_Cy3_ch00_MIP 27746

File:PpConcTest 03242014.JPG