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==sequencing library construction (2008-07-31)== ==2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 == reaction system x4 x3 H2O 43.2ul 172.8ul 518.4 2x Master mix 50ul 200ul 600 dUTP(1mM) 2ul 8ul 24 AmpF6.3(10uM) 2ul 8ul 24 AmpR6.3(10uM) 2ul 8ul 24 50x SYBG I 0.4ul 1.6ul 4.8 template(18.4ng/ul) 0.1ul 0.4ul 1.2 Total 100ul 400ul 2400 94C 3min -> 13cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C Bioneer column purification. Yield:167ng/ul x60ul ==step2. Digestion with MmeI== dUTP 250uM,167.3ng/ul x2 Total 20ul DNA 6ul 12 10X NEBuffer 4 2ul 4 1mM SAM(fresh) 2ul 4 2U/ul Mme I 8ul 16 ddH2O 2ul 4 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h MinElute column purify. Elute in 22ul EB. [[Image:20080801_after MmeI.jpg]]20080801_after MmeI ==step3. USER digestion== x2 DNA 10ul 20ul USER 3ul 6ul total 13ul 37C 12h. ==step4. S1 nuclease digestion== x2 10 x S1 nuclease buffer: 2ul 4ul DNA after USER digestion: 13ul 26ul S1 nuclease (10U/ul): 1ul 2ul ddH2O 4ul 8ul 37C 10mins. Minelute cloumn purify. Elute in 18ul H2O. [[Image:20080801_after USER and S1.jpg]]20080801_after USER and S1 ==step5. end repair== positive control Total 25ul H2O 13ul DNA 15ul 2ul(20080801_spacer_107bp) dNTP 2.5ul 2.5ul dATP 2.5ul 2.5ul 10xendrepair buffer 2.5ul 2.5ul enzyme 0.5ul 0.5ul extra ATP(10mM) 2.5ul 2.5ul Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O. ==step6. adapter ligation== total 30ul positive control negative control DNA 13ul 13ul ddH2O 13ul 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul 100uM Solexa_2 adaptor 0.5ul 0.5ul 0.5ul 2xQuickLiage buffer 15ul 15ul 15ul QuickLigase enzyme(NEB) 1ul 1ul 1ul extra ATP(10mM) 2.5ul 2.5ul 2.5ul Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul H2O. [[Image: 20080801_after endrepair and ligation.jpg]] PAGE selection 150-175bp. Ethanol precipitation. Elute in 15ul ddH2O. ==step7. Nick-translation== set up the ligation system: Total 20ul DNA 15ul 10x ThermoPol buffer 2ul 10mM dNTP 0.4ul 1mg/ml BSA 2ul Bst polymerase(8U/ul) 1ul 65C for 25 minutes -> keep on ice. ==step8. PCR of sequencig library== x3 Nick-translated DNA 10ul Solexa_PCR_up(10uM) 2ul 6ul Solexa_PCR_lo(10uM) 2ul 6ul 2xiProof master mix 50ul 150ul 50x SYBG 0.8ul 2.4ul ddH2O 35.2ul 106.6ul 98C 30sec -> 12 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold. [[Image:20080803_sequencing library.jpg]]20080803_sequencing library Purify with Qiaquick column. Elute in 30ul EB. yield: 22.9ng/ul(173nM)
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