Matt:LabNotes/2014-3-31

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Revision as of 00:31, 1 April 2014 by >Mzcai (Created page with "==Padlock Probe Concentration Test using Med Conc (100nM) Fixed Template== * Previously fixed 1nM template resulted in almost no rolonies *96-wel...")
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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 20 hours

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