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=Second Try: in tube SplintR Test with formamide and ET SSB= *[[Matt:LabNotes/2017-4-22|First try showed SplintR with 10% formamide had the best sensitivity and specificity of the conditions tried]] **Will try varying formamide concentration around 10% **Will qPCR with lower starting conc to get more accurate measurement ** ==Test Conditions== #Standard: SplintR only #SplintR + 5% formamide #SplintR + 7.5% formamide #SplintR + 10% formamide #SplintR + 12.5% formamide #SplintR + 15% formamide #SplintR + 10% formamide + 250ng ET SSB #Positive Control: Ampligase *For each test conditions have **one sample with ALL padlock probes and template ***Should see amplification **one sample with all padlock probes with NO MALAT1 template ***Should not see amplification ==Padlock Probes and Template== *ppCUX2 *ppBCL11B *ppRELN_1 *ppGFAP *ppMALAT1 **/5Phos/TTTCTGCCTTTACTTATCAATTCCTTCAGCTTCCCGATATCCGACGGTCTACTTCGTCGCGTCAGACCAAATGGAGGTATGACATATAATCT *Template for ppMALAT1: MALAT1_template **/5AmMC6/GAATTGATAAGTAAAGGCAGAAA AGATTATATGTCATACCTCCAT ==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="96" | Condition | width="51" | 10nM PP + Template | width="51" | 10X Buffer | width="51" | Formamide | width="51" | H2O | width="51" | Total |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 1 | SplintR | 1.5 or 1.8 (0.3ul of each 1uM oligo) | align="right" | 3 | align="right" | 0 | 25.5 or 25.2 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 2 | SplintR + 5% formamide | 1.5 or 1.8 (0.3ul of each 1uM oligo) | align="right" | 3 | align="right" | 1.5 | 24 or 23.7 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 3 | SplintR + 7.5% formamide | 1.5 or 1.8 (0.3ul of each 1uM oligo) | align="right" | 3 | align="right" | 2.25 | 23.25 or 22.95 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 4 | SplintR + 10% formamide | 1.5 or 1.8 (0.3ul of each 1uM oligo) | align="right" | 3 | align="right" | 3 | 22.5 or 22.2 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 5 | SplintR + 12.5% formamide | 1.5 or 1.8 (0.3ul of each 1uM oligo) | align="right" | 3 | align="right" | 3.75 | 21.75 or 21.45 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 6 | SplintR + 15% formamide | 1.5 or 1.8 (0.3ul of each 1uM oligo) | align="right" | 3 | align="right" | 4.5 | 21 or 20.7 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 7 | SplintR + 10% formamide + ET SSB | 1.5 or 1.8 (0.3ul of each 1uM oligo) | align="right" | 3 | align="right" | 3 | 22.5 or 22.2 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 8 | Ampligase | 1.5 or 1.8 (0.3ul of each 1uM oligo) | align="right" | 3 | align="right" | 0 | 25.5 or 25.2 | align="right" | 30 |} #Combine padlock probes and template in 1X Ligase buffer and possibly formamide #Add mineral oil on top #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 #Add 3ul SplintR Mix and incubate 15min #*SplintR Mix: 27ul SplintR + 4.5ul 10X SplintR Buffer + 13.5ul H2O #*Also add 0.4ul ET SSB to sample 7 #Incubate at 94C for 10min #Put all samples on ice and add 2ul Exo I/III mix #Incubate at 37C for 1.5hr #Incubate at 94C for 10min #qPCR all 16 samples {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''16X Volume''' |- | Captured template||5||0 |- | 10uM ISB_CA_AF||0.4||6.4 |- | 10uM ISB_CA_AR.T1||0.4||6.4 |- | 2X KAPA SYBG MM||12.5||200 |- | H2O||6.7||107.2 |- | Total||25||320 |} *Aliquot 20ul from 16X master mix and add 5ul captured template Program 98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min ==Results== [[Media:20170510_qPCR_PPcapture_SplintRformamideETSSB.xlsx|raw data here]]<br> [[File:20170510_qPCR_PPcapture_SplintRformamideETSSB.PNG|650px]] *Only Ampligase stands out from the rest [[File:20170510_qPCR_PPcapture_SplintRformamideETSSB_ScatterPlot.PNG|450px]] *12.5% formamide has the best sensitivity and seperation from NTC (specificity) *BUT 0%-10% formamide should all have higher sensitivity... *These results don't make sense **First try repeating PCR with triplicates and lower conc. (maybe Zymo column purify first) **Then try repeating experiment with concentrations that match actual conc. (100-200nM total) ==Repeat qPCR== #qPCR all 16 samples 1ul each 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)x30 -> 72C 3min [[File:20170515_qPCR_PPcapture_SplintRformamideETSSB_triplicates_ScatterPlot.PNG|450px]] *Full analysis here: [[Media:20170515_qPCR_PPcapture_SplintRformamideETSSB.xlsx]] ==Zymo column purify and Repeat qPCR== #Purify ssDNA from 20ul of 16 samples #Elute 10ul each #qPCR all 16 samples 1ul each 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)x30 -> 72C 3min [[File:20170515_qPCR_PPcapture_SplintRformamideETSSB_purified_ScatterPlot.PNG|450px]]
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