Matt:LabNotes/2014-4-9

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Efficiency of Ampligase in situ Test[edit]

  • 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