Matt:LabNotes/2014-4-15

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Efficiency of Ampligase in situ Test: Second Try

  • Previously results were not good enough to draw clear conclusion
  • General idea: Will use Exo I & Exo III OR Exo III only to digest non-ligased padlock probes, then use dcProbe2RevComp to hybridize to padlock probe and detect
    • Exo III only because Exo I will degrade all rolonies, leaving the padlock probe (even after ligation) nothing to attach to so it'll be washed away and not detected
  • Ligation with Ampligase should protect the padlock probes from digestion, hence comparing wells +Exo with those -Exo will show what percentage of padlock probes are ligated

Hybridize 10nM ppMALAT1

  • Add 100ul 10nM ppMALAT1 for 21.5hr @45C in 1X Ampligase buffer +/- 0.5U/ul Ampligase

A5&D2: -Ampligase

Component Volume
10X Ampligase Buffer 20
1uM ppMALAT1 2
H2O 178
Total 200

D3,D4,&D5: + Ampligase

Component Volume
10X Ampligase Buffer 30
1uM ppMALAT1 3
5 U/ul Ampligase 30
H2O 237
Total 300