Matt:LabNotes/2014-4-9
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Efficiency of Ampligase in situ Test[edit]
- Samples are wells F3-F10 from Matt:LabNotes/2014-3-31
- 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[edit]
- 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[edit]
- Wash with 2X SSC once
- Add 50ul Exonuclease mix
- Incubate @37C for 2 hrs
Exo I/III: F4 & F8[edit]
Component | Volume |
10X Exo III Buffer | 10 |
Exo I | 5 |
Exo III | 5 |
H2O | 80 |
Total | 100 |
Exo III: F3 & F9[edit]
Component | Volume |
10X Exo III Buffer | 10 |
Exo III | 5 |
H2O | 85 |
Total | 100 |
Control: F5, F6, F7 & F10[edit]
Component | Volume |
10X Exo III Buffer | 20 |
H2O | 180 |
Total | 200 |
Detect[edit]
- 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 4-10-2014
Results[edit]
Position | Well | Ampligase | Digestion | Hybridized Padlock Probes | Rolonies | % |
5 | F3 | - | Exo III | 203 | 15,805 | 1.28 |
6 | F4 | - | Exo I/III | 146 | 7,253 | 2.01 |
7 | F5 | - | - | 302 | 7,754 | 3.89 |
8 | F6 | - | - | 230 | 6,210 | 3.70 |
9 | F7 | + | - | 71 | 14,548 | 0.49 |
10 | F8 | + | Exo I/III | 0 | 13,323 | 0.00 |
11 | F9 | + | Exo III | 174 | 27,904 | 0.62 |
12 | F10 | + | - | 195 | 22,660 | 0.86 |
- Using bkg_mult = 3 because a lot of features weren't detected as "polonies"
- Also summed the intensity values of each pixel in an image since it should correlate more with number of tagged padlock probes rather than # of rolonies tagged by padlock probes
Position | Well | Ampligase | Digestion | Hybridized Padlock Probes | Rolonies | % | Intensity*Area |
5 | F3 | - | Exo III | 251 | 15,805 | 1.59 | 34,553,526 |
6 | F4 | - | Exo I/III | 191 | 7,253 | 2.63 | 24,825,840 |
7 | F5 | - | - | 311 | 7,754 | 4.01 | 53,719,236 |
8 | F6 | - | - | 310 | 6,210 | 4.99 | 47,879,730 |
9 | F7 | + | - | 107 | 14,548 | 0.74 | 31,531,795 |
10 | F8 | + | Exo I/III | 0 | 13,323 | 0.00 | 19,118,820 |
11 | F9 | + | Exo III | 222 | 27,904 | 0.80 | 42,094,484 |
12 | F10 | + | - | 245 | 22,660 | 1.08 | 49,782,492 |
File:20140410 EfficiencyAmpligaseinsituTest.JPG
Conclusion[edit]
- Exonuclease digestion did not seem complete
- Possibly adding 2uM of ppMALAT1 is too much for Exo I/III to handle in 2hrs? but doubt it
- Use 10nM ppMALAT1 next time to match the Exo I/III test
- Because -Ampligase wells with +Exo still detected signal, cannot draw any conclusions about +Ampligase wells
- The average % of detected rolonies of +Ampligase wells is less than average of -Ampligase wells:
- Does Ampligase decrease the efficiency % of hybridization?
- Or is it because the +Ampligase wells had more rolonies to start with?
- If that were true, it would imply ppMALAT1 is limiting reagent, which is unlikely given 2uM
- Try adding 50% glycerol in -Ampligase wells next time to better mimic +Ampligase wells