Matt:LabNotes/2014-3-26

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Revision as of 23:57, 26 March 2014 by >Mzcai (→‎1nM (D4))
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Padlock Probe Concentration Test using Low Conc (1nM) Fixed Template

  • Previously fixed 10uM template resulted in more targets than realistically would be in cells
  • 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 2X SSC to 85C (6ul 100nM MALAT1_Template + 60ul 20X SSC + 534ul H2O)
  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
    • Wrapped in parafilm and put in ziploc with wet towel

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