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==3 Genes Secondary Rolonies Mattek Dish== *Hosuk got much more primary rolonies (incl. ACTB and RAB7A) by culturing PGP1F without EGF ([[Hosuk:LabNotes/2014-3-3]]) *Since [[Matt:LabNotes/2014-2-25#Detection]] resulted in many MALAT1 secondary rolonies but few ACTB or RAB7A **This is possibly due to sample having very few ACTB or RAB7A **Also possible the dyes we got from Illumina (dcProbe0-Cy3 and dcProbe1-Cy3) have somehow degraded since MALAT1 detection was using a new dye-probe from IDT ===Procedure=== *Using Mattek dish from [[Hosuk:LabNotes/2014-3-3]] that was fixed in 4% formalin #Strip off dye-probes Hosuk used with 80% formamide pre-heated to 75C and incubated @45C for 15min #Wash with 1X PBS twice #Anneal Padlock Probes @45C for 24hrs (Put in at noon, some bubbles and hard to keep on middle glass) #*1X Ampligase Buffer #*2uM of each padlock probe #*0.5 U/ul Ampligase {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||20 |- | 200uM ppMALAT1||2 |- | 200uM ppACTB||2 |- | 200uM ppRAB7A||2 |- | 5U/ul Ampligase||20 |- | H2O||154 |- | Total||200 |} ====Secondary RCA==== #Aspirate but don't wash #'''Pre-annealing the FISSEQ_ppRCA primer''' for the rolling circle amplification reaction. ##1ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 199ul of '''2xSSC/30% formamide''' ##Preheat primer at 60C (Tm = 55C when '''not''' in 30% formamide) ##Add 200ul 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. ##171 ul ddH2O, ##20 ul 10x Phi29 buffer, ##2 ul 25 mM dNTP, ##4 ul 2 mM aminoallyl dUTP ##3 ul Phi29 DNA polymerase (Epicentre, low concentration) ##*200ul Total Volume #Add and incubate 20hrs at 30C. (Put in at 1pm 3/5/14) #Wash 1X PBS once #Add 200ul BS(PEG)9 mix to each well and incubate @RT for 1hr #Wash 1X PBS twice #Add 200ul 1M Tris pH 8.0 @RT for 1hr #Wash 1X PBS twice ====Detection==== *Make 100ul of 1uM dcProbe0-Cy3, dcProbe1-Cy3, and dcProbe2-Cy3 in 30% formamide and 2X SSC **dcProbe0 and dcProbe1 are from Illumina, dcProbe2 is from IDT ***Need to order more dcProbe1-Cy3 *Checked dcProbe0 *Strip *Checked dcProbe1 *Strip *Checked dcProbe2 **Dust fell into dish? Lots of non-specific binding so can't see secondary rolonies anymore ===Results=== *Using PISA7 Paramters: Gaussian std (3), upper bound (-2e-4), area upper (50), area lower (4), axratio lower (.6), circ upper (1.6), circ lower (.8), perim conn (8), bkgmult lower (7) '''dcProbe0''' *ACTB 2nd Rolonies **Pos1: 108 **Pos2: {| {{table}} | align="center" style="background:#f0f0f0;"|'''Gain 680''' | align="center" style="background:#f0f0f0;"|'''Gain 620''' |- | 130 2nd Rolonies||244 2nd Rolonies |- | [[File:Pos2_noEGF_dcProbe0_680_Cy3_ch00_MIP_PISA7Overlay.png|450px]]||[[File:Pos2_noEGF_dcProbe0_620_Cy3_ch00_MIP_PISA7Overlay.png|450px]] |} '''dcProbe1''' *RAB7A 2nd Rolonies **Pos2: {| {{table}} | align="center" style="background:#f0f0f0;"|'''Gain 680''' | align="center" style="background:#f0f0f0;"|'''Gain 620''' |- | 595 2nd Rolonies||779 2nd Rolonies |- | [[File:Pos2_noEGF_dcProbe1_680_Cy3_ch00_MIP_PISA7Overlay.png|450px]]||[[File:Pos2_noEGF_dcProbe1_620_Cy3_ch00_MIP_PISA7Overlay.png|450px]] |} '''dcProbe2''' *Didn't analyze because of dust ===Conclusion=== *The goal was to see if we could get ACTB and RAB7A 2nd Rolonies **Goal was successful ***There is no problem with dye-probes from Illumina or ppACTB/ppRAB7A ***[[Matt:LabNotes/2014-2-25#Experimental_Sample_.28E6_.26_F6.29|Previously]] the lack of ACTB and RAB7A 2nd Rolonies must have been due to low amount of mRNA in fixed sample
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