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==Padlock Probe Concentration Test using Fixed Template== *96-well plate NCI-H1975 Cells (C8, C9, C10, D8, D9, D10, E8, E9, E10, F9) **Alan already marked locations with good cell density *Test concentrations NegCtrl-0pM, 10pM, 50pM, 100pM, 10nM **2 wells for each ===Fixing Template in cells=== #Pre-heat 10uM MALAT1_Template in 2X SSC to 85C (50ul 100uM MALAT1_Template + 50ul 20X SSC + 400ul H2O) #Add 50ul to each well #*Incubate @RT for 15min #Aspirate but don't wash #Add 50ul BS(PEG)9 mix (10ul BS(PEG)9 + 490ul 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 24 hours (Started at 4pm) **Wrapped in parafilm and put in ziploc with wet towel ====Negative Control 0pM (C8 & C9)==== {| {{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 (D8 & D9)==== {| {{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 (E8 & E9)==== {| {{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 (C10 & D10)==== {| {{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 |} ====10nM (E10 & F9)==== {| {{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. ##2.5ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 497.5ul 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. ##427.5 ul ddH2O, ##50 ul 10x Phi29 buffer, ##5 ul 25 mM dNTP, ##10 ul 2 mM aminoallyl dUTP ##7.5 ul Phi29 DNA polymerase (Epicentre, low concentration) ##*500ul Total Volume #Add 50ul each well and incubate 20hrs at 30C. (Put in at 4:30pm 3/20/14) #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 500ul of 1uM dcProbe2-Cy3 (495ul 2XSSC & 30%formmaide + 5ul 100uM dcProbe2-Cy3) *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 3-24-2014''' ===Results=== *All imaging was done with 5% Laser and Gain of 550 **C10_100pM had a Z-thickness of ~37 microns so tried taking both 20 and 30 z-slices ***As seen in the table it didn't make a big difference *The disparity between C10 and D10 (both 100pM) can be partly attributed to different number of cells in field of view {| {{table}} | align="center" style="background:#f0f0f0;"|'''C10''' | align="center" style="background:#f0f0f0;"|'''D10''' |- | [[File:C10_100pM_G550_Cy3_z12_ch01.png|450px]]||[[File:D10_100pM_G550_30slices_Cy3_z09_ch01.png|450px]] |} {| {{table}} | align="center" style="background:#f0f0f0;"|'''Image''' | align="center" style="background:#f0f0f0;"|'''Rolony Count''' |- | C8_NegCtrl_G550_Cy3_ch00_MIP||0 |- | C9_NegCtrl_G550_Cy3_ch00_MIP||0 |- | D8_10pM_G550_Cy3_ch00_MIP||51 |- | D9_10pM_G550_Cy3_ch00_MIP||101 |- | E8_50pM_G550_Cy3_ch00_MIP||355 |- | E9_50pM_G550_Cy3_ch00_MIP||764 |- | C10_100pM_G550_Cy3_ch00_MIP||3216 |- | D10_100pM_G550_30slices_Cy3_ch00_MIP||521 |- | D10_100pM_G550_Cy3_ch00_MIP||497 |- | E10_10nM_G550_Cy3_ch00_MIP||17509 |- | F9_10nM_G550_Cy3_ch00_MIP||27746 |} [[File:PpConcTest_03242014.JPG]]
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