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===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 |}
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