Matt:LabNotes/2014-3-31: Difference between revisions

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===Detection===
===Detection===
*Made 600ul of 1uM dcProbe2-Cy3 (594ul 2XSSC & 30%formmaide + 6ul 100uM dcProbe2-Cy3)
*Made 650ul of 1uM dcProbe2-Cy3 (643.5ul 2XSSC & 30%formmaide + 6.5ul 100uM dcProbe2-Cy3)
**Extra volume since some always evaporates when heated
*Pre-heated @75C for 5 min and then added 50ul to each well
*Pre-heated @75C for 5 min and then added 50ul to each well
*Let sit @RT for 5 min
*Let sit @RT for 5 min

Revision as of 23:03, 2 April 2014

Padlock Probe Concentration Test using Med Conc (100nM) Fixed Template

  • Previously fixed 1nM template resulted in almost no rolonies
  • 96-well plate PGP1f Cells
    • Fixed 12/26/13 and RT with 12/30/13
    • Using wells C7-C10, F3-F10
      • Marked 1 position for each well (adapter clips on bottom&right)
      • .maf file saved in 3-31-2014
  • Test padlock probe concentrations NegCtrl-0pM, 10pM, 50pM, 100pM, 1nM, 10nM

Fixing Template in cells

  1. Pre-heat 100nM MALAT1_Template to 85C
  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
  7. Wash with 1X PBS twice

Anneal Padlock Probes

  • 1X Ampligase Buffer
  • 0.5 U/ul Ampligase
  • Incubate @ 45C for 21 hours

Negative Control 0pM (C7&C8)

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

10pM (C9&C10)

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

50pM (F3&F4)

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

100pM (F5&F6)

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

1nM (F7&F8)

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

10nM (F9&F10)

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 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. 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 each well and incubate 20hrs at 30C.
  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

  • Made 650ul of 1uM dcProbe2-Cy3 (643.5ul 2XSSC & 30%formmaide + 6.5ul 100uM dcProbe2-Cy3)
    • Extra volume since some always evaporates when heated
  • 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 4-2-2014
    • Imaged with 5% laser and Gain of 550