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===Day 2=== #Wash with 1X PBS once #*FISSEQ Dish has very little solution left... but not bone dry #Add 200ul BS(PEG)9 (20ul BS(PEG)9 + 980ul 1X PBS) and incubate 1hr at RT #*Opened a new BS(PEG)9, couldn't see any solution in the vial but added 465ul DMSO just in case #*'''THERE WAS NOTHING IN THE VIAL! AFTER 465ul DMSO ADDED FINAL VOLUME 465 WHEN SHOULD BE 565 AND NOT VISCOUS LIKE NORMAL''' #Wash with 1X PBS twice #Add Tris pH8.0 and incubate 30min at RT #Wash with 1X PBS twice #RNA Removal #*Add 200ul RNase Mix (100ul RNase H 10X Buffer + 10ul Riboshredder + 50ul RNase H + 840ul H2O) and incubate 1hr at 37C #Wash with H2O twice #To DARTFISH slide: Add 100nM Padlock Probe Mix with 1000X suppv2 oligos in one sample and only 100nM Padlock Probe Mix in other and incubate ~1hr at 60C as oven slowly decreases to 55C and held for another ~20hr #*Put in pipette tip box with bottom filled with water and parafilmed around edges of box {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | DEPC-H2O||33.43 |- | Ampligase Buffer||10 |- | Justin's Batch 4 7/13/2015 Padlock Probes 559nM]]||18 |- | 100nM suppv2 Oligos || 28.57 |- | Ampligase||10 |- | Total||100 |} {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | DEPC-H2O||62 |- | Ampligase Buffer||10 |- | Justin's Batch 4 7/13/2015 Padlock Probes 559nM]]||18 |- | Ampligase||10 |- | Total||100 |} #To FISSEQ slide and dish: Add 200ul CircLigaseII mix and incuabate 1hr at 60C #*Put in pipette tip box with bottom filled with water and parafilmed around edges of box {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | DEPC-H2O||384 |- | CircLigase Buffer 10X||60 |- | MnCl2 50mM||30 |- | Betaine 5M|| 120 |- | CircLigase II (100U/ul)||6 |- | Total||600 |} #Wash FISSEQ samples with PBS and store in PBS at 4C overnight
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