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===V8 Padlock Probe Capture=== *Dilute 1.5ul of 1ug/ul UHRR into 30ul total(50ng/ul final conc) *DNA is 12878 80.3ng/ul *V8 padlock probes: [[Matt:LabNotes/2015-1-21|874nM]] **Add 5.2ul to samples 3 & 6 and then dilute remaining ~4ul to 19ul final volume {| 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" | V8 (42.7ng/ul) | width="51" | V8 (8.19ng/ul) | width="51" | 10X SplintR Buffer (*=Ampligase) | width="51" | H2O | width="51" | Total |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 1 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 1 | align="right" | 3 | align="right" | 26 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 2 | align="right" | 0 | align="right" | 0 | align="right" | 0 | align="right" | 5 | align="right" | 3 | align="right" | 22 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 5.2 | align="right" | 0 | align="right" | 3 | align="right" | 21.8 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 4 | align="right" | 0.6 | align="right" | 0 | align="right" | 0 | align="right" | 1 | align="right" | 3 | align="right" | 25.4 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 5 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 5 | align="right" | 3 | align="right" | 19 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 6 | align="right" | 16.4 | align="right" | 0 | align="right" | 5.2 | align="right" | 0 | align="right" | 3 | align="right" | 5.4 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 7 | align="right" | 0 | align="right" | 3.8 | align="right" | 0 | align="right" | 2 | 3* | align="right" | 21.2 | align="right" | 30 |- style="font-size:11pt" valign="bottom" | align="right" height="14" | 8 | align="right" | 3 | align="right" | 0 | align="right" | 0 | align="right" | 5 | 3* | align="right" | 19 | 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 prepared on ice! **Samples 7-8: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O **Samples 1-6: 12ul SplintR + 2ul 10X SplintR Buffer + 6ul H2O *Incubate at 37C for 15min *94C for 2min *Aliquot 10ul from each into separate strip of tubes and put at 4C for 1 hr until qPCR *Enzyme digest template of remaining mixture 37C for 1 hr **1ul Exo I/III (1:1) for DNA **1ul RNaseH and Riboshredder (1:1) for RNA ***First add 1ul 5M NaCl (100nM NaCl or KCl for Riboshredder) *94C for 2min *Add 1ul Exo I/III (1:1) for unligated padlock probes to ALL tubes *37C for 1 hr *94C for 2min
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