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==Secondary Rolony Generation== Samples: [[Matt:LabNotes/2014-2-21]] ===Procedure=== ====Anneal Padlock Probes==== *1X Ampligase Buffer *2uM of each padlock probe *0.5 U/ul Ampligase *Incubate @ 45C for 24 hours **Wrapped in parafilm and put in ziploc with wet towel =====Fixed Template - 25U Ampligase (C9 & F9)===== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 200uM ppMALAT1||1 |- | 5U/ul Ampligase||10 |- | H2O||79 |- | Total||100 |} =====Fixed Template - 50U Ampligase (C10 & F10)===== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 200uM ppMALAT1||1 |- | 5U/ul Ampligase||20 |- | H2O||69 |- | Total||100 |} =====1st Rolony (E6 & F6)===== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 200uM ppMALAT1||1 |- | 200uM ppACTB||1 |- | 200uM ppRAB7A||1 |- | 5U/ul Ampligase||10 |- | H2O||77 |- | Total||100 |} =====Empty Negative Control (D7 & E7)===== *Not going to waste Ampligase since it's highly unlikely anything will be ligated, but will do RCA in these wells =====Pre-circularized Padlock Probes (C7 & C8)===== *Combined all the pre-circle ppMALAT1 made on: [[Matt:LabNotes/2014-2-10]] into 64ul *75% EtOH for 10 minutes *Wash with 1X PBS 2 times *0.1N HCl for 2 minutes *Wash with 1X PBS 3 times *Add 32ul to C7 & C8 =====Fixed Pre-circularized Padlock Probes (F7 & F8)===== *Not going to do anything during Ampligase step ====Secondary RCA==== #Aspirate each well but don't wash #*F7 well was dried up #'''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 to each well, 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 to each well and incubate overnight at 30C. (Put in at 7pm 2/26/14) #Wash 1X PBS once #Add 50ul BS(PEG)9 mix to each well and incubate @RT for 1hr (12ul BS(PEG)9 + 588ul 1X PBS) #Wash 1X PBS twice #Add 50ul 1M Tris pH 8.0 @RT for 1hr #Wash 1X PBS twice ====Detection==== *To all wells add 50ul 1uM dcProbe2-Cy3 (6ul 100uM dcProbe2-Cy3 + 180ul 100% formamide + 60ul 20X SSC + 354ul H2O preheated to 75C) **Strip E6 & F6 with 80% formamide @ 37C *To E6 & F6 add 50ul 1uM dcProbe0-Cy3 (10ul 10uM dcProbe0-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C) **Strip with 80% formamide @37C *To E6 & F6 add 50ul 1uM dcProbe1-Cy3 (10ul 10uM dcProbe1-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C) *'''Images saved under 2-27-2014 folder''' ===Results=== *Whole experiment summarized in powerpoint: [[File:FridayZhangMeeting_140228.pptx]] ====Positive Control - Fixed Template (F9 & F10)==== *Comparing # of Fixed Template with # of 2nd Rolonies Generated **F9 used 0.5U/ul Ampligase while F10 used 1U/ul **When using MALAT1_1stRolonyFISH to count Fixed Templates, only counted large clumps of targets while single targets may not be bright enough to see {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''# Fixed Template''' | align="center" style="background:#f0f0f0;"|'''# 2nd Rolonies''' |- | F9 (0.5 U/ul)||30||5,624 |- | F10 (1 U/ul)||61||7,883 |} *Secondary Rolonies were many! Looks Great! **Here is F9 example PISA7 counted (red marks counted rolonies) image while raw tif can be found in powerpoint [[File:F9_dcProbe2_G550_Cy3_ch00_MIP_PISA7Overlay.png | 450px]] ====Ampligase Concentration Comparison: 0.5U/ul vs 1U/ul==== *Looking at # of 2nd Rolonies Generated and assuming about same # of fixed template in each **C9 & F9 are 0.5U/ul **C10 & F10 are 1U/ul *No significant difference so will keep using 0.5U/ul {| {{table}} | align="center" style="background:#f0f0f0;"|'''Ampligase Conc''' | align="center" style="background:#f0f0f0;"|'''0.5 U/ul''' | align="center" style="background:#f0f0f0;"|'''1 U/ul''' |- | Row C||5,532||4,536 |- | Row F||5,624||7,883 |} ====Positive Control - Fixed Precircularized ppMALAT1_NH2 (F7 & F8)==== *It was hard to tell if any was fixed in the first place with dcProbe2RevComp-Cy3 *2nd Rolony results seem to confirm that since there was less 2nd Rolonies than even the negative control {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''# Fixed Circle''' | align="center" style="background:#f0f0f0;"|'''# 2nd Rolonies''' |- | F7||9 (high gain)||5 |- | F8||12 (high gain)||11 |} ====Positive Control - Not Fixed Precircularized ppMALAT1 (C7 & C8)==== *C8 had ~5-10X more secondary rolonies than C7 but both were way more than what we've seen before **No point actually counting since there's nothing to compare to *Example of C8 [[File:C8_dcProbe2_G550_Cy3_ch00_MIP.png | 450px]] ====Negative Control (D7 & E7)==== *Saw more signal than many 2nd Rolony Ampligase tests from before so should do negative control every time in the future to get a baseline of false positives *D7: 43 *E7: 28 ====Experimental Sample (E6 & F6)==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''E6 Targets''' | align="center" style="background:#f0f0f0;"|'''E6 2nd Rolonies''' | align="center" style="background:#f0f0f0;"|'''F6 Targets''' | align="center" style="background:#f0f0f0;"|'''F6 2nd Rolonies''' |- | ACTB||6||28||6||13 |- | RAB7A||7||46||6||15 |- | MALAT1||5||246||4||145 |} *Based on the MALAT1 results it seems that trying to quantify targets with 1stRolonyFISH probes isn't very accurate *Since the 2nd Rolonies of ACTB and RAB7A are in the range of Negative control we can't confidently say any ACTB or RAB7A padlock probes produced 2nd Rolonies *It's curious that only MALAT1 seemed successful **Could be that dcProbe2-Cy3 (recently ordered from IDT to replace the old dye from Illumina) which was used to detect MALAT1 works better **Could also be MALAT1 is more abundant than the other 2, which is what was reported in Harvard's FISSEQ Science paper
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