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==Experiment== ===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 ===qPCR=== ====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 |} ====PCR Test==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''4X Volume''' |- | Captured template||1||0 |- | 10uM ISB_CA_AF||0.4||1.6 |- | 10uM ISB_CA_AR.T1||0.4||1.6 |- | 2X KAPA SYBG MM||12.5||50 |- | H2O||10.7||42.8 |- | Total||25||96 |} *Aliquot 24ul from 4X master mix and add 1ul captured template Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x26 -> 72C 3min [[File:20161119_CA12kOct2016V4_invitroCapture_PCRtest.JPG|450px]] ====PCR==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1X Volume''' | align="center" style="background:#f0f0f0;"|'''3.5X Volume''' |- | Captured template||12||0 |- | 10uM Forward Primer||2||7 |- | 10uM Reverse Primer||2||0 |- | 2X KAPA SYBG MM||50||175 |- | H2O||34||119 |- | Total||100||301 |} *Indx1:T4 *Indx2:SplintR *Indx3:Ampligase *Aliquot 86ul from 3.5X master mix and add 12ul captured template and 2ul corresponding reverse primer Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min [[File:20161203_invitro_CA12kOct2016V4_PCR.png|450px]] ====AMPure Bead Purification==== *1:1 beads to amplicon volume ratio **Eluted with 50ul total each sample ====PAGE Check==== *Load 2ul sample + 2ul loading dye [[File:2016-12-15_CA12kOct2016_V4_invitroCapture_Library.jpg|250px]] ====Sequencing Library==== MC20161215_CA12kOct20161215_V4_T4_Indx1<br> MC20161215_CA12kOct20161215_V4_SplintR_Indx2<br> MC20161215_CA12kOct20161215_V4_Amp_Indx3
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