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==Protocol== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:11pt" valign="bottom" | width="51" height="14" | Sample # | width="122" | Condition | width="51" | 30nM PP + Template | width="51" | 10X Buffer | width="51" | Formamide | width="51" | DMF | width="51" | Betaine | width="51" | DMSO | width="80" | H2O | width="51" | Total |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 1 | SplintR | 4.5 or 5.4 (0.9ul of each 1uM oligo) | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 0 | 22.5 or 21.6 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 2 | SplintR + 5% formamide | 4.5 or 5.4 (0.9ul of each 1uM oligo) | align="right" | 3 | align="right" | 1.5 | align="right" | 0 | align="right" | 0 | align="right" | 0 | 21 or 20.1 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 3 | SplintR + 10% formamide | 4.5 or 5.4 (0.9ul of each 1uM oligo) | align="right" | 3 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 0 | 19.5 or 18.6 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 4 | SplintR + 15% formamide | 4.5 or 5.4 (0.9ul of each 1uM oligo) | align="right" | 3 | align="right" | 4.5 | align="right" | 0 | align="right" | 0 | align="right" | 0 | 18 or 17.1 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 5 | SplintR + 10% DMF | 4.5 or 5.4 (0.9ul of each 1uM oligo) | align="right" | 3 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | 19.5 or 18.6 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 6 | SplintR + 1M Betaine | 4.5 or 5.4 (0.9ul of each 1uM oligo) | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 6 | align="right" | 0 | 16.5 or 15.6 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 7 | SplintR + 5% DMSO | 4.5 or 5.4 (0.9ul of each 1uM oligo) | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 1.5 | 21 or 20.1 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 8 | Ampligase | 4.5 or 5.4 (0.9ul of each 1uM oligo) | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 0 | 22.5 or 21.6 | align="right" | 30 |} #Combine padlock probes and template in 1X Ligase buffer and possibly additives #Add mineral oil on top #Incubate at 95C for 3min #Incubate at 55C for 18hr #To sample 8 add 3ul Ampligase Mix and incubate at 55C for 1hr30min #*Ampligase Mix: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O #Move samples 1-7 to 37C #*Move one tube at a time and add enzyme after holding tube between fingers for 5 sec #Add 3ul SplintR Mix and incubate 15min #*SplintR Mix: 27ul SplintR + 4.5ul 10X SplintR Buffer + 13.5ul H2O #Incubate at 94C for 5min #Put all samples on ice and add 2ul Exo I/III mix #Incubate at 37C for 1hr #Incubate at 94C for 5min #Purify 20ul with Zymo ssDNA purification kit, elute 20ul #Dilute 5ul in 45ul H2O (10x dilution) #qPCR all 16 samples with triplicates {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''48X Volume''' |- | Captured template||1||0 |- | 10uM ISB_CA_AF||0.4||19.2 |- | 10uM ISB_CA_AR.T2||0.4||19.2 |- | 2X KAPA SYBG MM||12.5||600 |- | H2O||10.7||513.6 |- | Total||25||1,152 |} *Aliquot 24ul from 48X master mix and add 1ul captured template Program 98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min ===Results=== [[Media:20170610_qPCR_PPcapture_SplintRformamide.xlsx|raw data here]]<br> [[File:20170610_qPCR_PPcapture_SplintRformamide_ScatterPlot.PNG|450px]]
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