Matt:LabNotes/2014-4-5

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Revision as of 22:59, 7 April 2014 by >Mzcai (→‎Detection)
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Padlock Probe Concentration Test using Low Conc (1nM) in H2O Fixed Template

  • Previously fixed 100nM in H2O (as opposed to 2XSSC) and ended up with too many templates as determined by number of rolonies generated
  • Sample was previously used to fix 1nM in 2X SSC which was too low
    • Assuming no template was fixed and 2 extra rounds of BS(PEG)9 has no effect we can re-use this sample
  • 24-well plate PGP1f Cells cultured w/o EGF
    • Fixed 3/21/14 and RT 3/21/14
    • Using wells A4, A5, D2, D3, D4, D5
      • Marked 2 positions for each well (adapter clips on bottom&right)
      • .maf file saved in 3-26-2014
  • Test padlock probe concentrations NegCtrl-0pM, 10pM, 50pM, 100pM, 1nM, 10nM

Fixing Template in cells

  1. Pre-heat 1nM MALAT1_Template in H2O to 85C
  2. Add 100ul to each well
    • Incubate @RT for 15min
  3. Aspirate but don't wash
  4. Add 100ul 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
  7. Wash with 1X PBS twice

Anneal Padlock Probes

  • 1X Ampligase Buffer
  • 0.5 U/ul Ampligase
  • Incubate @ 45C for 24 hours (Put in at 5:30pm)

Negative Control 0pM (A4)

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

10pM (A5)

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

50pM (D2)

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

100pM (D3)

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

1nM (D4)

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

10nM (D5)

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

Secondary RCA

  1. Aspirate 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 100ul 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. 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 100ul each well and incubate 20hrs at 30C. (Put in at 6:00pm)
  5. Wash 1X PBS once
  6. Add 100ul BS(PEG)9 mix to each well and incubate @RT for 1hr
  7. Wash 1X PBS twice
  8. Add 100ul 1M Tris pH 8.0 @RT for 30min
  9. Wash 1X PBS twice

Detection

  • Made 600ul of 1uM dcProbe2-Cy3 (594ul 2XSSC & 30%formmaide + 6ul 100uM dcProbe2-Cy3)
  • Pre-heated @75C for 5 min and then added 100ul to each well
  • Let sit @RT for 5 min
  • Washed twice with 2X SSC
  • Saved in 4-7-2014

Results

  • Using Gain of 550
Image pp Concentration [pM] Rolonies
'Pos1_NegCtrl_Cy3_ch00_MIP.tif' 0 5963
'Pos2_NegCtrl_Cy3_ch00_MIP.tif' 0 9467
'Pos3_10pM_Cy3_ch00_MIP.tif' 10 9439
'Pos4_10pM_Cy3_ch00_MIP.tif' 10 6314
'Pos5_50pM_Cy3_ch00_MIP.tif' 50 6502
'Pos6_50pM_Cy3_ch00_MIP.tif' 50 6789
'Pos7_100pM_Cy3_ch00_MIP.tif' 100 6967
'Pos8_100pM_Cy3_ch00_MIP.tif' 100 4796
'Pos11_10nM_Cy3_ch00_MIP.tif' 10000 2167
'Pos12_10nM_Cy3_ch00_MIP.tif' 10000 6316
  • Using Gain of 650
Image pp Concentration [pM] Rolonies
'Pos3_10pM_G650_Cy3_ch00_MIP.tif' 10 6310
'Pos4_10pM_G650_Cy3_ch00_MIP.tif' 10 6494
'Pos5_50pM_G650v2_Cy3_ch00_MIP.tif' 50 6789
'Pos6_50pM_G650_Cy3_ch00_MIP.tif' 50 6971
'Pos7_100pM_G650_Cy3_ch00_MIP.tif' 100 4796
'Pos8_100pM_G650_Cy3_ch00_MIP.tif' 100 4986
'Pos10_1nM_G650_Cy3_ch00_MIP.tif' 1000 2164
'Pos11_10nM_G650_Cy3_ch00_MIP.tif' 10000 6321
'Pos12_10nM_G650_Cy3_ch00_MIP.tif' 10000 5962

Conclusion

  • None of the sample showed significant difference from negative control so can only assume template concentration was too low
  • Unlike last time where negative control had absolutely zero rolonies, the negative control showed a LOT of dim signal
    • The rolonies counted here had a raw intensity value of ~1,000
    • The true rolonies counted last time had a raw intensity value of ~10,000
  • The other samples were also dim rolonies with intensity of ~1,000 instead of the normal ~10,000
  • What can cause dim signal?
    • Autofluorescence
      • But would expect to see this last time too unless difference between PGP1f and NCI-H1975
    • Dye binding to fixed template
      • Wouldn't expect to see so many since template concentration was low
      • Also should've shown up in negative control last time too