Matt:LabNotes/2014-4-9

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Revision as of 23:42, 10 April 2014 by >Mzcai (→‎Hybridize 2uM ppMALAT1)
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Efficiency of Ampligase in situ Test

  • 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 it's possible 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 2uM ppMALAT1

  • Add 50ul 2uM ppMALAT1 for 21.5hr @45C (7:00pm-4:30pm ) in 1X Ampligase buffer +/- 0.5U/ul Ampligase

F3-F6: -Ampligase

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

F7-F10: + Ampligase

Component Volume
10X Ampligase Buffer 20
200uM ppMALAT1 2
5 U/ul Ampligase 20
H2O 158
Total 200

Digestion

  • Wash with 2X SSC once
  • Add 50ul Exonuclease mix

Exo I/III: F4 & F8

Component Volume
10X Exo III Buffer 10
Exo I 5
Exo III 5
H2O 80
Total 100

Exo III: F3 & F9

Component Volume
10X Exo III Buffer 10
Exo III 5
H2O 85
Total 100

Control: F5, F6, F7 & F10

Component Volume
10X Exo III Buffer 20
H2O 180
Total 200

Detect

  • Wash with 2X SSC twice
  • Add 50ul 1uM dcProbe2RevComp-Cy3 (ATTO 488 dye)
    • Pre-heated to 75C then snap-cooled before adding
  • Wash with 2X SSC twice
  • Images saved in 4-9-2014

Results

Conclusion