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===June 26, 2013=== * Library prep for Quartz-seq samples A1 and A2: ** Adapter ligation using KAPA Rapid Ligation, USER, and PCR with KAPA SYBR Fast (See Jeff and Noi's protocols). I used NEBNext Index Primers (the loop adapters). The following is directly from the NEBNext Multiplex Oligos for Illumina manual. {| {{table}} | align="center" style="background:#f0f0f0;"|'''Product''' | align="center" style="background:#f0f0f0;"|'''Index Primer Sequence''' | align="center" style="background:#f0f0f0;"|'''Expected Index Primer Sequence Read''' |- | NEBNext Index 11 Primer for Illumina||5Β΄-CAAGCAGAAGACGGCATACGAGAT[GTAGCC]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3Β΄||GGCTAC |- | NEBNext Index 12 Primer for Illumina||5Β΄-CAAGCAGAAGACGGCATACGAGAT[TACAAG]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3Β΄||CTTGTA |- | |} I used Index 12 for sample A1 (Terra) and Index 11 for Sample A2 (KAPA HiFi Uracil+). ====CORE fragmentation and library prep==== * Work in PCR hood * UV treat H2O, tips, tubes * Purified Quartz WTA products using Ampure XP beads ** Added 100 ul beads to 107 ul product ** Eluted in 14.2 ul H2O * UDG/Endo IV rxn 14.2 ul template 1 ul 1:121 UDG 1 ul 1:121 Endo IV 1.8 ul Exo- buffer ------------------ Total = 18 ul # 2 hrs @ 37 degC # 15 min @ 65 degC # Hold @ 4 degc ----------------------------------- * Nick translation 18 ul UDG/Endo IV rxn 1 ul 1:50 Exo- 1 ul 1:25 1 mM dNTP (40 uM) --------------------------- Total = 20 ul # 1 hrs @ 37 degC # 15 min @ 75 degC # Hold @ 4 degC ----------------------------------- * Ligation rxn 20 ul nick translation rxn 25 ul 2X KAPA ligation buffer 1.67 ul 1.5 uM loop adapter 1.33 ul H2O KAPA DNA ligase ----------------------------- Total = 50 ul # 15 min @ 20 degC # Add 2 ul USER # 15 min @ 37 degC # Hold @ 4 degC ----------------------------------- * Ampure XP bead purification ** 50 ul beads ** Elute in 20 ul H2O * PCR 20 ul template 1 ul 10 uM PCR_F 1 ul 10 uM NEBNext index 3 ul H2O 2X KAPA SYBR mix ------------------------ Total = 50 ul # 30 sec @ 98 degC # 15 cycles: ## 10 sec @ 98 degC ## 30 sec @ 65 degC ## 45 sec @ 72 degC # 2 min @ 72 degC # Hold @ 4 degC ------------------------------------- [[File: 2013_06_26 quartz seq 500 pg uhrr.jpd|400px]] I size selected the KAPA library (Index 11) and gave it to Alan for sequencing ([[Arichard:Samples/Sequencing_samples|AR_QC_500pgUhrrKapa_Jun26]]).
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