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==Test Exo I/III Digestion in situ== *Sample wells E10 & F9 from [[Matt:LabNotes/2014-3-19]] **E10 + Exo I/III **F9 - Exo I/III ===Hybridize 10nM ppMALAT1=== *Add 50ul 10nM ppMALAT1 for 20hr in 1X Ampligase buffer (7:00pm-3:00pm) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 1uM ppMALAT1||1 |- | H2O||89 |- | Total||100 |} ===Exo I/III Digestion=== #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=== *Add 50ul 1uM dcProbe2RevComp-Cy3 (ATTO488 dye) **Pre-heat to 75C *Wash with 2X SSC twice *Images saved in '''4-7-2014''' ===Results=== *With Laser 5% and Gain 500 couldn't detect any padlock probes in either well *With Laser 5% and Gain 550 could detect {| {{table}} | align="center" style="background:#f0f0f0;"|'''E10: +Exo''' | align="center" style="background:#f0f0f0;"|'''F9: -Exo''' |- | 18 Padlock Probes||389 Padlock Probes |- | [[File:E10_Exo%2B_L5_G550_488_ch00_MIP_PISA7Overlay.png | 650px]]||[[File:F9_Exo-_L5_G550_488_ch00_MIP_PISA7Overlay.png | 650px]] |} ===Conclusion=== *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
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