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===V4 Padlock Probe Capture=== *Dilute 1.5ul of 1ug/ul UHRR into 30ul total(50ng/ul final conc) *DNA is 12878 80.3ng/ul *V4 padlock probes: *[[Matt:LabNotes/2016-11-2|20.7nM]] {| 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="51" | RNA (50ng/ul) | width="51" | DNA (80.3ng/ul) | width="51" | V4 | width="51" | 10X Buffer | width="51" | H2O | width="51" | Total |- style="font-size:11pt" valign="bottom" | height="14" | NTC | align="right" | 0 | align="right" | 0 | align="right" | 10 | align="right" | 3 | align="right" | 17 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | height="14" | SplintR | align="right" | 3 | align="right" | 0 | align="right" | 10 | align="right" | 3 | align="right" | 14 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | height="14" | T4 | align="right" | 3 | align="right" | 0 | align="right" | 10 | align="right" | 3 | align="right" | 14 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | height="14" | Ampligase | align="right" | 0 | align="right" | 2 | align="right" | 10 | align="right" | 3 | align="right" | 15 | align="right" | 30 |} *Added 50ul mineral oil on top '''Program'''<br> * 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h * -> add 3ul Enzyme mix *For Ampligase: -> 55C 20h -> 94C 2min -> add 2ul Exo I/III -> 37C 2h -> 94C 2min -> 4C hold *For SplintR and T4: -> 37C 15min -> 94C 2min -> add 2ul Exo I/III -> 37C 2h -> 94C 2min -> 4C hold *Add 3ul Enzyme mix prepared on ice! **Ampligase: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O **NTC & SplintR: 12ul SplintR + 2ul 10X SplintR Buffer + 6ul H2O **T4: 1ul T4 DNA Ligase + 1ul 10X T4 Buffer + 5.5ul ATP + 2.5ul H2O ***Thermo T4 DNA Ligase HC, 30 Weiss U/ul ***ATP 10mM
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