Matt:LabNotes/2014-4-15

From ZhangLabWiki
Jump to navigation Jump to search

Efficiency of Ampligase in situ Test: Second Try[edit]

  • 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[edit]

  • 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[edit]

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

Exo I/III: D3[edit]

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

Exo III: D2&D4[edit]

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

Control: A5&D5[edit]

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

Detect[edit]

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

Results[edit]

Ampligase Exo Digest Image Name Rolonies Detected with dcProbe2RevComp Rolonies Detected with Cy3 Before Digestion Sum of dcProbe2RevComp Intensity
-Amp Ctrl A5_Pos3_L5_G550_488_ch00_MIP 32 568 37,259,522.00
-Amp Ctrl A5_Pos4_L5_G550_488_ch00_MIP 0 448 25,703,163.00
-Amp Exo III D2_Pos5_L5_G550_488_ch00_MIP 0 415 20,739,052.00
-Amp Exo III D2_Pos6_L5_G550_488_ch00_MIP 0 339 25,296,778.00
-Amp Exo I/III D3_Pos7_L5_G550_488_ch00_MIP 29 395 47,514,945.00
-Amp Exo I/III D3_Pos8_L5_G550_488_ch00_MIP 0 365 21,611,171.00
+Amp Exo III D4_Pos9_L5_G550_488_ch00_MIP 0 1065 28,048,612.00
+Amp Exo III D4_Pos10_L5_G550_488_ch00_MIP 0 2027 30,495,676.00
+Amp Ctrl D5_Pos11_L5_G550_488_ch00_MIP 43 2760 57,266,371.00
+Amp Ctrl D5_Pos12_L5_G550_488_ch00_MIP 97 3012 47,420,054.00
  • No clear conclusion can be drawn, especially because of A5_Pos4 (Control) showing no signal when there should be and D3_Pos7 showing signal when there shouldn't be
    • If those were switched the results would look much better
  • If we treat those two as anomalies then:
    • The average Sum Intensity of Neg Ctrl = 22,549,000
    • The average Sum Intensity of Pos Ctrl = 47,315,315
  • The average Sum Intensity of Experimental (+Amp, Exo III) = 29,272,144
 (29-22)/(47-22) = 28%
  • This "rough" calculation estimates that 28% of padlock probes are ligated

Rolonies Stable[edit]

Image Name Rolonies Detected Before Rolonies Detected After
A5_Pos3_L5_G550_Cy3_ch00_MIP 345 568
A5_Pos4_L5_G550_Cy3_ch00_MIP 543 448
D5_Pos11_L5_G550_Cy3_ch00_MIP 2504 2760
D5_Pos12_L5_G550_Cy3_ch00_MIP 3880 3012
  • No significant change in rolonies detected with dcProbe2-Cy3