Matt:LabNotes/2014-4-15
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Efficiency of Ampligase in situ Test: Second Try[edit]
- Previously results were not good enough to draw clear conclusion
- Samples are wells A5, D2, D3, D4, D5 from Matt:LabNotes/2014-4-5
- 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