Matt:LabNotes/2014-4-6
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Test Exo I/III Digestion in situ[edit]
- Sample wells E10 & F9 from Matt:LabNotes/2014-3-19
- E10 + Exo I/III
- F9 - Exo I/III
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]
- Wash with 2X SSC once
- Add 50ul Exo I/III to each well (2.5ul 20U/ul Exo I and 2.5ul 200U/ul Exo III)
- Exo I/III mix: 5ul 10X Exo III Buffer + 2.5ul Exo I + 2.5ul Exo III + 40ul H2O
- Positive control: 50ul of (5ul 10X Exo III Buffer + 45ul H2O)
- Incubate 37C for 2hrs
- 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