Matt:LabNotes/2014-3-19

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Revision as of 22:19, 19 March 2014 by >Mzcai
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Padlock Probe Concentration Test using Fixed Template

  • 96-well plate NCI-H1975 Cells (C8, C9, C10, D8, D9, D10, E8, E9, E10, F9)
    • Alan already marked locations with good cell density
  • Test concentrations NegCtrl-0pM, 10pM, 50pM, 100pM, 10nM
    • 2 wells for each

Fixing Template in cells

  1. Pre-heat 10uM MALAT1_Template in 2X SSC to 85C (50ul 100uM MALAT1_Template + 50ul 20X SSC + 400ul H2O)
  2. Add 50ul to each well
    • Incubate @RT for 15min
  3. Aspirate but don't wash
  4. Add 50ul BS(PEG)9 mix (10ul BS(PEG)9 + 490ul 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 (Started at 4pm)
    • Wrapped in parafilm and put in ziploc with wet towel

Negative Control 0pM (C8 & C9)

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

10pM (D8 & D9)

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

50pM (E8 & E9)

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

100pM (C10 & D10)

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

10nM (E10 & F9)

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