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==Padlock Probe Concentration Test using Med Conc (100nM) Fixed Template== *[[Matt:LabNotes/2014-3-26 | Previously]] fixed 1nM template resulted in almost no rolonies *96-well plate PGP1f Cells **Fixed 12/26/13 and RT with 12/30/13 **Using wells C7-C10, F3-F10 ***Marked 1 position for each well (adapter clips on bottom&right) ***.maf file saved in 3-31-2014 *Test padlock probe concentrations NegCtrl-0pM, 10pM, 50pM, 100pM, 1nM, 10nM ===Fixing Template in cells=== #Pre-heat 100nM MALAT1_Template to 85C #Add 50ul to each well #*Incubate @RT for 15min #Aspirate but don't wash #Add 50ul BS(PEG)9 mix (12ul BS(PEG)9 + 588ul 1X PBS) #*Incubate @RT for 1hr #Wash with 1X PBS twice #Add 50ul 1M Tris pH 8.0 #* Incubate @ RT for 30min #Wash with 1X PBS twice ===Anneal Padlock Probes=== *1X Ampligase Buffer *0.5 U/ul Ampligase *Incubate @ 45C for 21 hours ====Negative Control 0pM (C7&C8)==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 5U/ul Ampligase||10 |- | H2O||80 |- | Total||100 |} ====10pM (C9&C10)==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 1nM ppMALAT1||1 |- | 5U/ul Ampligase||10 |- | H2O||79 |- | Total||100 |} ====50pM (F3&F4)==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 1nM ppMALAT1||5 |- | 5U/ul Ampligase||10 |- | H2O||75 |- | Total||100 |} ====100pM (F5&F6)==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 1nM ppMALAT1||10 |- | 5U/ul Ampligase||10 |- | H2O||70 |- | Total||100 |} ====1nM (F7&F8)==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 10nM ppMALAT1||10 |- | 5U/ul Ampligase||10 |- | H2O||70 |- | Total||100 |} ====10nM (F9&F10)==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 1uM ppMALAT1||1 |- | 5U/ul Ampligase||10 |- | H2O||79 |- | Total||100 |} ===Secondary RCA=== #Aspirate but don't wash #'''Pre-annealing the FISSEQ_ppRCA primer''' for the rolling circle amplification reaction. ##3ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 597ul of '''2xSSC/30% formamide''' ##Preheat primer at 60C (Tm = 55C when '''not''' in 30% formamide) ##Add 50ul and incubate at 45C for 15min. ##Aspirate, and wash using 2x SSC twice ##Aspirate, and wash using 0.1x SSC twice #Prepare the rolling circle amplification reaction mixtures on ice. ##513 ul ddH2O, ##60 ul 10x Phi29 buffer, ##6 ul 25 mM dNTP, ##12 ul 2 mM aminoallyl dUTP ##9 ul Phi29 DNA polymerase (Epicentre, low concentration) ##*600ul Total Volume #Add 50ul each well and incubate 20hrs at 30C. #Wash 1X PBS once #Add 50ul BS(PEG)9 mix to each well and incubate @RT for 1hr #Wash 1X PBS twice #Add 50ul 1M Tris pH 8.0 @RT for 30min #Wash 1X PBS twice ===Detection=== *Made 650ul of 1uM dcProbe2-Cy3 (643.5ul 2XSSC & 30%formmaide + 6.5ul 100uM dcProbe2-Cy3) **Extra volume since some always evaporates when heated *Pre-heated @75C for 5 min and then added 50ul to each well *Let sit @RT for 5 min *Washed twice with 2X SSC *'''Saved in 4-2-2014''' ===Results=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Position''' | align="center" style="background:#f0f0f0;"|'''Well''' | align="center" style="background:#f0f0f0;"|'''Concentration''' | align="center" style="background:#f0f0f0;"|'''Rolony Count''' |- | Pos1||C7||0pM||0 |- | Pos2||C8||0pM||0 |- | Pos3||C9||10pM||2458 |- | Pos4||C10||10pM||1952 |- | Pos5||F3||50pM||15805 |- | Pos6||F4||50pM||7253 |- | Pos7||F5||100pM||7754 |- | Pos8||F6||100pM||6210 |- | Pos9||F7||1nM||14548 |- | Pos10||F8||1nM||13323 |- | Pos11||F9||10nM||27904 |- | Pos12||F10||10nM||22660 |} ====Compare these results with [[Matt:LabNotes/2014-3-19 | 10uM Template]]==== *In red: Previous concentration test where 10uM template in 2X SSC was fixed **Although a higher concentration, I think 2X SSC interferes with BS(PEG)9 fixation hence the actual lower concentration *In blue: This concentration test results [[File:PpConcTest_04042014.png]]
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