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=Agi15k_Feb2017 V4 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 | 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" | 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" | 23.4 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 3 | SplintR DNA | align="right" | 3.6 | align="right" | 3.8 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 19.6 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 4 | SplintR RNA | align="right" | 3.6 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 22.4 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 5 | SplintR NTC | align="right" | 3.6 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 0 | align="right" | 23.4 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 6 | SplintR 20% formamide DNA | align="right" | 3.6 | align="right" | 3.8 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 6 | align="right" | 13.6 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 7 | SplintR 20% formamide RNA | align="right" | 3.6 | align="right" | 0 | align="right" | 1 | align="right" | 0 | align="right" | 3 | align="right" | 6 | align="right" | 16.4 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 8 | SplintR 20% formamide NTC | align="right" | 3.6 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 3 | align="right" | 6 | align="right" | 17.4 | align="right" | 30 |} '''Program'''<br> *1,2,3,6: 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 16h *4,5,7,8: 55C 18h *Ampligase: add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer) *SplintR: add 3ul SplintR enzyme mix (13.5ul SplintR + 2.25ul 10X SplintR Buffer + 6.75ul H2O) *Ampligase: -> 55 C 20h -> 94C 2min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2min -> 4C hold *SplintR: -> 37 C 15min -> 94C 10min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2 min -> 4C hold *Zymo columns **Elute 10ul **Sample 2 was only able to pipette 20ul instead of 30ul of the ligation reaction so theoretically has 33% lower conc after elution from column ====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=== *On 5/18/2017 #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 *Average Ct values {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:11pt" valign="bottom" | align="right" width="51" height="14" | | width="51" | DNA | width="51" | RNA | width="51" | NTC |- style="font-size:11pt" valign="bottom" | height="14" | Ampligase | align="right" | 13.7 | N/A | align="right" | 27.1 |- style="font-size:11pt" valign="bottom" | height="14" | SplintR | align="right" | 12.1 | align="right" | 12.1 | align="right" | 12.5 |- style="font-size:11pt" valign="bottom" | height="14" | SplintR 20% Formamide | align="right" | 19.2 | align="right" | 18.9 | align="right" | 17.6 |} [[Media:20170518_Agi15kFeb17_invitrocapture_SplintR20formamide.xlsx|Excel data]] ==Conclusion== *Accidentally chose 20% Formamide, should have been lower *Odd that in 20% Formamide NTC had more captured padlock probes than the EXACT SAME conditions plus DNA or RNA
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