Matt:LabNotes/2014-4-15

From ZhangLabWiki
Revision as of 00:34, 17 April 2014 by >Mzcai (→‎Detect)
Jump to navigation Jump to search

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 24hr @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

Digestion

  • Wash with 2X SSC once
  • Add 100ul Exonuclease mix to each well
  • Incubate @37C for 3 hrs

Exo I/III: D3

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

Exo III: D2&D4

Component Volume
10X Exo III Buffer 20
Exo III 10
H2O 170
Total 200

Control: A5&D5

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

Detect

  • Wash with 2X SSC twice
  • Add 100ul 1uM dcProbe2RevComp-Cy3 (ATTO 488 dye)
    • Pre-heated to 75C
  • Wash with 2X SSC twice