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==Efficiency of Ampligase in situ Test: Second Try== *[[Matt:LabNotes/2014-4-9 | Previously]] results were not good enough to draw clear conclusion *Samples are wells A5, D2, D3, D4, D5 from [[Matt:LabNotes/2014-4-5]] **[[Matt:LabNotes/2014-4-5#Results | # of Rolonies at each position]] *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 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 10nM ppMALAT1=== *Add 100ul 10nM ppMALAT1 for 24hr @45C in 1X Ampligase buffer +/- 0.5U/ul Ampligase A5&D2: -Ampligase {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||20 |- | 1uM ppMALAT1||2 |- | H2O||178 |- | Total||200 |} D3,D4,&D5: + Ampligase {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||30 |- | 1uM ppMALAT1||3 |- | 5 U/ul Ampligase||30 |- | H2O||237 |- | Total||300 |} ===Digestion=== *Wash with 2X SSC once *Add 100ul Exonuclease mix to each well *Incubate @37C for 3 hrs ====Exo I/III: D3==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Exo III Buffer||10 |- | Exo I||5 |- | Exo III||5 |- | H2O||80 |- | Total||100 |} ====Exo III: D2&D4==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Exo III Buffer||20 |- | Exo III||10 |- | H2O||170 |- | Total||200 |} ====Control: A5&D5==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Exo III Buffer||20 |- | H2O||180 |- | Total||200 |} ===Detect=== *Wash with 2X SSC twice *Add 100ul 1uM dcProbe2RevComp-Cy3 (ATTO 488 dye) **Pre-heated to 75C *Wash with 2X SSC twice ===Results=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Ampligase''' | align="center" style="background:#f0f0f0;"|'''Exo Digest''' | align="center" style="background:#f0f0f0;"|'''Image Name''' | align="center" style="background:#f0f0f0;"|'''Rolonies Detected with dcProbe2RevComp''' | align="center" style="background:#f0f0f0;"|'''Rolonies Detected with Cy3 Before Digestion''' | align="center" style="background:#f0f0f0;"|'''Sum of dcProbe2RevComp Intensity''' |- | -Amp||Ctrl||A5_Pos3_L5_G550_488_ch00_MIP||32||568|| 37,259,522.00 |- | -Amp||Ctrl||A5_Pos4_L5_G550_488_ch00_MIP||0||448|| 25,703,163.00 |- | -Amp||Exo III||D2_Pos5_L5_G550_488_ch00_MIP||0||415|| 20,739,052.00 |- | -Amp||Exo III||D2_Pos6_L5_G550_488_ch00_MIP||0||339|| 25,296,778.00 |- | -Amp||Exo I/III||D3_Pos7_L5_G550_488_ch00_MIP||29||395|| 47,514,945.00 |- | -Amp||Exo I/III||D3_Pos8_L5_G550_488_ch00_MIP||0||365|| 21,611,171.00 |- | +Amp||Exo III||D4_Pos9_L5_G550_488_ch00_MIP||0||1065|| 28,048,612.00 |- | +Amp||Exo III||D4_Pos10_L5_G550_488_ch00_MIP||0||2027|| 30,495,676.00 |- | +Amp||Ctrl||D5_Pos11_L5_G550_488_ch00_MIP||43||2760|| 57,266,371.00 |- | +Amp||Ctrl||D5_Pos12_L5_G550_488_ch00_MIP||97||3012|| 47,420,054.00 |} *No clear conclusion can be drawn, especially because of A5_Pos4 (Control) showing no signal when there should be and D3_Pos7 showing signal when there shouldn't be **If those were switched the results would look much better *If we treat those two as anomalies then: **The average Sum Intensity of Neg Ctrl = 22,549,000 **The average Sum Intensity of Pos Ctrl = 47,315,315 *The average Sum Intensity of Experimental (+Amp, Exo III) = 29,272,144 (29-22)/(47-22) = 28% *This "rough" calculation estimates that 28% of padlock probes are ligated ====Rolonies Stable==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Image Name''' | align="center" style="background:#f0f0f0;"|'''Rolonies Detected Before''' | align="center" style="background:#f0f0f0;"|'''Rolonies Detected After''' |- | A5_Pos3_L5_G550_Cy3_ch00_MIP||345||568 |- | A5_Pos4_L5_G550_Cy3_ch00_MIP||543||448 |- | D5_Pos11_L5_G550_Cy3_ch00_MIP||2504||2760 |- | D5_Pos12_L5_G550_Cy3_ch00_MIP||3880||3012 |} *No significant change in rolonies detected with dcProbe2-Cy3
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