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===Day 2=== #Wash with 1X PBS once #Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT #Wash with 1X PBS twice #Add 1M Tris pH 8.0 and incubate 30min at RT #Wash with 1X PBS twice #RNA Removal #*Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C #Wash with nf-H2O twice ====To Sample 3 Only==== #Add 50ul gel casting mix '''filtered and degassed''' #*5% gel with 125uM Acrydite-Amine linker, 5mM BS(PEG)9, and Use [[Matt:LabNotes/2016-7-26#Qubit_ssDNA | 478nM TB12kApr2016 V4 Probes]] instead of H2O (33.5ul) #Seal in plastic bag and vacuum out air before filling with argon #Let sit at RT for 30min #Aspirate non-polymerized gel and wash once with 1M Tris #Prepare Ampligase mix on ice #*Preheat Padlock Probes then snap cool before adding Ampligase buffer and enzyme {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Ampligase Buffer||10 |- | [[Matt:LabNotes/2016-7-26#Qubit_ssDNA | 478nM]]||80 |- | Ampligase||10 |- | Total||100 |} #Add mix to sample and incubate for 30min at 37C #Move sample to 60C and oven slowly decreases to 55C and held for ~24hr ====To Samples 1 & 2==== #Prepare Ampligase mix on ice #*Preheat Padlock Probes then snap cool before adding Ampligase buffer and enzyme {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | Ampligase Buffer||10 |- | [[Matt:LabNotes/2016-7-26#Qubit_ssDNA | 478nM]]||80 |- | Ampligase||10 |- | Total||100 |} #Add mix to sample and incubate for 30min at 37C #Move sample to 60C and oven slowly decreases to 55C and held for ~44hr
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