AlanFung:LabNotes/Rolony/2014-2-19

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Optimum Annealing Time for padlock probes[edit]

  • Hosuk and Matt reported that they have tried 1 hour vs 4 hours annealing time and they see a difference in the quantity of signals
  • In the experiment we will be trying 1 hour vs 4 hours vs overnight annealing incubation to figure out the optimum time for incubation and wether an overnight incubation will help with generating more signals.

Annealing duration to test[edit]

  • 1 hour
  • 4 hours
  • 24 hours

Setup[edit]

  • In this experiment I will be using 4 mattek dish containing PGP1F P11 cells prepared by Hosuk on 11/15/2013 fixed on 11/19/2013

Fixing Template in cells[edit]

  1. Pre-heat 10uM MALAT1_Template in 2X SSC to 85C (30ul 100uM MALAT1_Template + 30ul 20X SSC + 240ul H2O) - Stored in Hosuk 4C non-dye box
    • Add 100ul to each mattek dish, Incubate @RT for 15min
  2. Wash with 1X PBS
  3. Add 100ul BS(PEG)9 mix (6ul BS(PEG)9 + 294ul 1X PBS)
    • Incubate @RT for 1hr
  4. Wash with 1X PBS twice
  5. Add 100ul 1M Tris pH 8.0
    • Incubate @ RT for 30min

Annealing Padlock Probes[edit]

  1. Add 100ul 2uM ppMALAT1 in 2X SSC @45C for 1hour, 4 hour and 24 hours starts at 3:15pm
  2. Wash 2X with 2X SSC
  3. Prepare dye for 3 scans (30ul 20X SSC, 90ul 100% formamide, 177ul H2O, 3ul Dye)
  4. Preheat dye at 75C for 5 mins
  5. Add 100ul 1uM dcProbe2RevComp-Cy3 in 2X SSC + 30% formamide
  6. Incubate at RT for 10 mins
  7. Wash 2 2X SSC twice

Confocal Microscopy[edit]

  1. To detect dcProbe2RevComp-Cy3 use seq_488.seq(it also saves smart gain, smart offset and pinhole)
  2. Strip w preheat 80% formamide on the stage for 15 mins

Imaging[edit]

  • 10-15um, 10 z stack image, lower limit begin go up till upper limit end
  • resolution (2kx2k)
  • 16bit,600Hz
  • when done save file (experiment_dish(mattekdate)_step#_dyeprobe_channel)
  • export as Tif and quite save experiment (lif)

Results[edit]

  • From the images pretty much all the signals observed are from background noise.
  • Based on the imaging results, we learnt that storing the hybridized padlock probes overnight in 4C will not preserve the annealed padlock probes and since we are not seeing any signals from the dcprobe2revcomp-cy3 we can conclude that the padlock probes somehow denatured and become denatured form the template.
  • We learnt that it is best to perform imaging right after the hybridization of padloack probes
  • I will be repeating the experiment using the same cross-linked template, I can start off by stripping off all the padlock probes and rehybridize new ones onto template

Strip off and Re-hybridize with ppMALAT1[edit]

  1. Strip all 3 mattek dish w preheated (75C) 80% formamide for 15 mins at RT
  2. Wash 2X with 2X SSC
  3. Add 200ul 2uM ppMALAT1 (100ul more than usual to avoid evaporation, also wrap dish with parafilm) in 2X SSC @45C for 1hour, 4 hour and 24 hours starts at 11:05am
  4. Wash 2X with 2X SSC
  5. Prepare dye for 3 scans (30ul 20X SSC, 90ul 100% formamide, 177ul H2O, 3ul Dye)
  6. Preheat dye at 75C for 5 mins
  7. Add 100ul 1uM dcProbe2RevComp-Cy3 in 2X SSC + 30% formamide
  8. Incubate at RT for 10 mins
  9. Wash w 1X PBS twice
  10. Imaging

Strip off and Re-hybridize with MALAT1_1stRolonyFish[edit]

  1. Strip on stage w preheated (75C) 200ul 80% formamide for 15 mins at RT
  2. Wash 2X with 2X SSC
  3. Add 100ul preheated (75C) 1uM MALAT1_1stronlonyfish in 2X SSC + 30% formamide
  4. Incubate at RT for 10 mins
  5. Wash w 1x PBS twice
  6. Image

Results[edit]

1hr Annealing Time[edit]

  • A:dcprobe
  • B:MALAT1_1stRolonyFish

File:MIP ppannealingtime PGP1F 1hr.jpg

4hr Annealing Time[edit]

  • C:dcprobe
  • D:MALAT1_1stRolonyFish

File:MIP ppannealingtime PGP1F 4hr.jpg

24hr Annealing Time[edit]

  • E:dcprobe
  • F:MALAT1_1stRolonyFish

File:MIP ppannealingtime PGP1F 24hr.jpg