AlanFung:LabNotes/Rolony/2014-3-5

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Secondary Rolony Generation for Padlock Probe Concentration Test[edit]

Samples[edit]

  • 96 well plate containing NCIH-1975 cells fixed by Hosuk
  • Location and save location of 12 position in 96 well plate for the experiment
  • High Concentration (100pM)
  • Low Concentration (1pM)
  • Ultra Low Concentration (0.1pM)

Fixing Template in cells[edit]

  1. Pre-heat 10uM MALAT1_Template in 2X SSC to 85C (60ul 100uM MALAT1_Template + 60ul 20X SSC + 480ul H2O)
  2. Add 50ul to each well
    • Incubate @RT for 15min
  3. Aspirate but don't wash
  4. Add 50ul BS(PEG)9 mix (12ul BS(PEG)9 + 588ul 1X PBS)
    • Incubate @RT for 1hr
  5. Wash with 1X PBS twice
  6. Add 50ul 1M Tris pH 8.0
    • Incubate @ RT for 30min

Dilute 2nM ppMALAT1[edit]

  • 100pM
  • 1pM
  • 0.1pM

Secondary RCA[edit]

  1. Aspirate each well but don't wash
  2. Pre-annealing the FISSEQ_ppRCA primer for the rolling circle amplification reaction.
    1. 3ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 597ul of 2xSSC/30% formamide
    2. Preheat primer at 60C (Tm = 55C when not in 30% formamide)
    3. Add 50ul to each well, 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. 513 ul ddH2O,
    2. 60 ul 10x Phi29 buffer,
    3. 6 ul 25 mM dNTP,
    4. 12 ul 2 mM aminoallyl dUTP
    5. 9 ul Phi29 DNA polymerase (Epicentre, low concentration)
      • 600ul Total Volume
  4. Add 50ul to each well and incubate overnight at 30C. (Put in at 7pm 2/26/14)
  5. Wash 1X PBS once
  6. Add 50ul BS(PEG)9 mix to each well and incubate @RT for 1hr (12ul BS(PEG)9 + 588ul 1X PBS)
  7. Wash 1X PBS twice
  8. Add 50ul 1M Tris pH 8.0 @RT for 1hr
  9. Wash 1X PBS twice

Anneal Padlock Probes for 24 hours[edit]

  1. Wash twice w PBS
  2. 1X Ampligase Buffer (10ul of stock 10X)
  3. Add padlock probe to each well with their corresponding concentration (35ul)
  4. 0.5 U/ul Ampligase (5ul of stock 5U/ul)
  5. Incubate @ 45C for 24 hours
  6. Wrapped in parafilm and put in ziploc with wet towel

Group 1 100pM ppMALAT1[edit]

Group 1-100pM   Group 2-1pM   Group 3-0.1pM
Image Well Mark & Find   Image Well Mark & Find   Image Well Mark & Find
A C3 1   E D3 2   I C5 4
B F8 15   F F5 14   J F6 7
C F3 13   G C6 9   K C7 10
D E6 8   H E7 11   L F7 12
  3 4 5 6 7 8
A            
B            
C C3   C5 C6 C7  
D D3          
E       E6 E7  
F F3   F5 F6 F7 F8
G            
H            

File:Ppconcentration NCHI1975 Group1.jpg

File:Ppconcentration NCHI1975 Group2.jpg

File:Ppconcentration NCHI1975 Group3.jpg