Matt:LabNotes/2014-4-6

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Test Exo I/III Digestion in situ[edit]

Hybridize 10nM ppMALAT1[edit]

  • Add 50ul 10nM ppMALAT1 for 20hr in 1X Ampligase buffer (7:00pm-3:00pm)
Component Volume
10X Ampligase Buffer 10
1uM ppMALAT1 1
H2O 89
Total 100

Exo I/III Digestion[edit]

  1. Wash with 2X SSC once
  2. Add 50ul Exo I/III to each well (2.5ul 20U/ul Exo I and 2.5ul 200U/ul Exo III)
    1. Exo I/III mix: 5ul 10X Exo III Buffer + 2.5ul Exo I + 2.5ul Exo III + 40ul H2O
    2. Positive control: 50ul of (5ul 10X Exo III Buffer + 45ul H2O)
    3. Incubate 37C for 2hrs
  3. Wash with 2X SSC twice

Detect with dcProbe2RevComp[edit]

  • Add 50ul 1uM dcProbe2RevComp-Cy3 (ATTO488 dye)
    • Pre-heat to 75C
  • Wash with 2X SSC twice
  • Images saved in 4-7-2014

Results[edit]

  • With Laser 5% and Gain 500 couldn't detect any padlock probes in either well
  • With Laser 5% and Gain 550 could detect
E10: +Exo F9: -Exo
18 Padlock Probes 389 Padlock Probes
File:E10 Exo+ L5 G550 488 ch00 MIP PISA7Overlay.png File:F9 Exo- L5 G550 488 ch00 MIP PISA7Overlay.png

Conclusion[edit]

  • Detecting hybridized padlock probes is low efficiency
  • Exonuclease I/III does digest DNA in situ because E10 had 17,000 rolonies and F9 had 28,000