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=Agi15k_Feb2017 V4 + 5% formamide in vitro Capture= ===Calculate Probes Needed=== ====V4==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe:target''' | align="center" style="background:#f0f0f0;"|'''1000:1''' | align="center" style="background:#f0f0f0;"|'''''' |- | Probe size||4998||probes |- | DNA templet||300||ng |- | gDNA MW||1.95x10^12||g/mol |- | gDNA (300ng) ||1.5385x10^-19||mol |- | Probe (1000:1)||1.5385x10^-16||mol |- | Probe MW (4998, 157nt)||2.387x10^8||g/mol |- | Amount Probe req'd||36.7||ng |} ===Probes, Target and Ampligase Buffer Mix=== *gDNA: 12878 (80.3ng/ul) *UHRR: 740000-41 (1ug/ul) *[[Matt:LabNotes/2017-2-8#Summary | Agi15k_Feb2017 Probe Design]] *[[Matt:LabNotes/2017-4-12 | V4 Probe Production (10.3 ng/ul)]] {| 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="134" | Condition | width="69" | V4 (10.3ng/ul) | width="106" | 12878 DNA (80.3ng/ul) | width="70" | UHRR (1ug/ul) | width="96" | Ampligase Buffer | width="90" | SplintR Buffer | width="60" | Formamide or DMF | width="90" | Ribolock 40U/ul | width="51" | H2O | width="51" | Total |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 1 | Ampligase DNA | align="right" | 3.6 | align="right" | 3.8 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 19.6 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 2 | Ampligase NTC | align="right" | 3.6 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 23.4 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 3 | SplintR 10% DMF DNA | align="right" | 3.6 | align="right" | 3.8 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 3 | align="right" | 0 | align="right" | 16.6 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 4 | SplintR 10% DMF RNA | align="right" | 3.6 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 3 | align="right" | 3 | align="right" | 0.5 | align="right" | 18.9 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 5 | SplintR 10% DMF NTC | align="right" | 3.6 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 3 | align="right" | 0 | align="right" | 20.4 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 6 | SplintR 5% formamide DNA | align="right" | 3.6 | align="right" | 3.8 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 1.5 | align="right" | 0 | align="right" | 18.1 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 7 | SplintR 5% formamide RNA | align="right" | 3.6 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 3 | align="right" | 1.5 | align="right" | 0.5 | align="right" | 20.4 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 8 | SplintR 5% formamide NTC | align="right" | 3.6 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 1.5 | align="right" | 0 | align="right" | 21.9 | align="right" | 30 |} '''Program'''<br> *95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 16h **Should have added RiboLock after reached 55C since it would be denatured at 95C *Ampligase: add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer) *SplintR: add 3ul SplintR enzyme mix (10ul SplintR + 2ul 10X SplintR Buffer + 4ul Ribolock + 4ul H2O) *Ampligase: -> 55 C 20h -> 94C 2min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2min -> 4C hold *SplintR: -> 37 C 30min -> 94C 10min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2 min -> 4C hold *Take 30ul and purify with Zymo columns **Left in 4C 1 hour after first column spin down **Elute 10ul ====AmpLigase enzyme mix==== {| class="wikitable" style="text-align:center;{{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Stock conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Final conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Prepare volume 10ul''' |- | AmpLigase||5||U/ul||0.5||U/ul||1.00 |- | 10x AmpLigase Buffer||10||x||1||x||1.00 |- | H2O||||||||||8.00 |- | Total||||||||||10.00 |} ===Quantify=== #qPCR all 8 samples 1ul each with triplicates {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''25X Volume''' |- | Captured template||1||0 |- | 10uM ISB_CA_AF||0.4||10 |- | 10uM ISB_CA_AR.T2||0.4||10 |- | 2X KAPA SYBG MM||12.5||312.5 |- | H2O||10.7||267.5 |- | Total||25||600 |} *Aliquot 24ul from 24X master mix and add 1ul captured template Program 98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x30 -> 72C 3min [[File:20170622_Agi15kFeb17_invitrocapture_SplintR5formamide.PNG|350px]][[File:20170622_Agi15kFeb17_invitrocapture_SplintR5formamide_Ct.PNG|250px]] *[[Media:20170622_Agi15kFeb17_invitrocapture_SplintR5formamide.xlsx|Excel Data]] *No separation between DNA, RNA, and NTC samples for SplintR *Sequence results to see if there's no difference really **Stop Ampligase NTC at 28 cycles **Stop others at 19 cycles ===Add Sequence Adapters PCR=== ====Primers==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Primer''' | align="center" style="background:#f0f0f0;"|'''Sequence''' | align="center" style="background:#f0f0f0;"|'''Index #''' |- | ISB_CA_AF||AATGATACGGCGACCACCGAGATCTACACGCCTGCATATCGGGAAGCTGAAG|| |- | ISB_CA_AR.T1||CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTCCCGATATCCGACGG||Indx1 |- | ISB_CA_AR.T2||CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG||Indx2 |- | ISB_CA_AR.T3||CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTCCCGATATCCGACGG||Indx3 |} {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Index''' | align="center" style="background:#f0f0f0;"|'''Forward Primer''' | align="center" style="background:#f0f0f0;"|'''Reverse Primer''' |- | 1||1||ISB_CA_AF||ISB_CA_AR.T1 |- | 2||2||ISB_CA_AF||ISB_CA_AR.T2 |- | 3||1||ISB_CA_AF||ISB_CA_AR.T1 |- | 4||2||ISB_CA_AF||ISB_CA_AR.T2 |- | 5||3||ISB_CA_AF||ISB_CA_AR.T3 |- | 6||1||ISB_CA_AF||ISB_CA_AR.T1 |- | 7||2||ISB_CA_AF||ISB_CA_AR.T2 |- | 8||3||ISB_CA_AF||ISB_CA_AR.T3 |} ====PCR==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''8.5X Volume''' |- | Captured template||5||0 |- | 10uM Forward Primer||2||17 |- | 10uM Reverse Primer||2||0 |- | 2X KAPA SYBG MM||50||425 |- | H2O||41||348.5 |- | Total||100||790.5 |} *Aliquot 93ul from 8.5X master mix and add 5ul captured template and 2ul corresponding reverse primer Program 98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x30 -> 72C 3min *Sample 1: Stopped at 20 cycles *Sample 2: Stopped at 28 cycles *Sample 3-5: Stopped at 18 cycles *Sample 6-8: Stopped at 17 cycles [[Media:20170626_Agi15kFeb17_invitrocapture_SplintR5formamide.xlsx|Excel data with PCR curve]] [[File:2017-07-10_Agi15kFeb17V4_invitroCapture_SeqLibrarySizeCheck.jpg|250px]] *Only Ampligase NTC band looks different, otherwise every sample has bands of expected size for captured padlock probes with sequencing adapters *Nanodrop: every sample was ~500ng/ul +/- 10ng/ul *Mix Samples 1&2, 3&4&5, 6&7&8 in equal ratios (20ul each) *Gel size selection 3 lanes **Load each well with 10ul mix (~5ug) + 10ul 6x loading buffer [[File:2017-06-27_Agi15kFeb17V4_invitroCapture_SeqLibrarySizeSelect.jpg|450px]] *Cut out band and shear through 0.5ul tube into 2.0ul tube *Add 450ul 1X TE Buffer and vortexed at 37C for 1hr *Centrifuged at 15,000 rpm for 3 min at RT *Transferred the clear spnt. to Nanosep columns and centrifuged at 15,000 rpm for 3 min *Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min *Transferred spnt to fresh 1.5 mL tube *Precipitated in 3 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2 *Vortexed and placed the 8 tubes at -80C 30min *Spun 8 tubes at 10,000rpm at 4C for 30min *Discard supernatant and add 650ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min *Discard supernatant and let dry in hood for 10min *Resuspend each tube with 10ul and combine ====For Sequencing==== *MC20170619_Agi15kFeb2017_V4_AmpLigase_Indx1,2 *MC20170619_Agi15kFeb2017_V4_SplintRDMF_Indx1,2,3 *MC20170619_Agi15kFeb2017_V4_SplintRForm_Indx1,2,3
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