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==sequencing library construction== ===2nd PCR amplification with AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 === reaction system x14 x2 H2O 42.6ul 596.4ul 2x Master mix 50ul 700ul dUTP(1mM) 2ul 28ul AmpF6.3(10uM) 2ul 28ul AmpR6.3(10uM) 2ul 28ul 50x SYBG I 0.4ul 5.6ul template(4ng/ul) 0.5ul 2 uleach Total 100ul 1400ul 94C 3min -> 9cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C Bioneer column purification. Yield: No_21:40.5ng/ul; No_22:39.1ng/ul; No_23:35.6ng/ul; No_24:32.4ng/ul; No_25:47ng/ul; No_26:35.2ng/ul; No_21_2: 28.9ng/ul; ===Digestion with MmeI=== No_21~26 x6 Total 40ul dUTP_PCR 20ul 10X NEBuffer 4 4ul 24 1mM SAM(fresh) 8ul 48 2U/ul Mme I 8ul 48 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h. MinEulte column purification. Elute in 12ul EB. ===step3. USER digestion=== DNA 10ul USER 3ul total 13ul 37C 2h. ===step4. S1 nuclease digestion=== No21~26 x7 10 x S1 nuclease buffer: 2ul 14ul DNA after USER digestion: 13ul 13ul S1 nuclease (10U/ul): 1ul 7ul ddH2O 4ul 28ul 37C 10mins. Minelute cloumn purify. Elute in 16ul H2O. ===step5. end repair=== No21~26 positive control x7 Total 25ul H2O 13ul DNA 15ul 2ul(20080801_spacer_107bp) dNTP 2.5ul 2.5ul 17.5 dATP 2.5ul 2.5ul 17.5 10xendrepair buffer 2.5ul 2.5ul 17.5 enzyme 0.5ul 0.5ul 3.5 extra ATP(10mM) 2.5ul 2.5ul 17.5 Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O. ===step6. adapter ligation=== No21~26 positive control negative control x8 DNA 13ul 13ul ddH2O 13ul 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul 4 100uM Solexa_2_PE adaptor 0.5ul 0.5ul 0.5ul 4 2xQuickLiage buffer 15ul 15ul 15ul 120 QuickLigase enzyme(NEB) 1ul 1ul 1ul 8 extra ATP(10mM) 2.5ul 2.5ul 2.5ul 20 Keep at room temperature (~25C) for 10 minutes. Purify with Minelute cilumn. Elute in 20ul EB. TBE gel size selection of ~175bp fragment. Elute in 15ul ddH2O. ===step7. Nick-translation=== set up the ligation system: x8 Total 20ul DNA 15ul 10x ThermoPol buffer 2ul 16ul 10mM dNTP 0.4ul 3.2ul 1mg/ml BSA 2ul 16ul Bst polymerase(8U/ul) 1ul 8ul 65C for 25 minutes -> keep on ice. ===step8. PCR of sequencig library=== x8 Nick-translated DNA 20ul Solexa_PCR_up(10uM) 2ul 16ul Solexa_PCR_PE_loH(10uM) 2ul 16ul 2xiProof master mix 50ul 400ul 50x SYBG 0.8ul 6.4ul ddH2O 25.2ul 201.6ul 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold. [[Image:20090130_parallel Solexa Sequencing library No21~26.jpg]]20090130_parallel Solexa Sequencing library No21~26 Qiaquick column purification and quantification: No_21 PGP1_iPS1: 16.4 ng/ul(90.3nM) x 30ul; No_22 PGP1_L: 14.1 ng/ul(77.6nM) x 30ul; No_23 PGP3_L: 14.7 ng/ul(80.9nM) x 30ul; No_24 PGP9_L: 17.6 ng/ul(96.9nM) x 30ul; No_25 PGP1_F.2: 18.5 ng/ul(93.4nM) x 30ul; No_26 PGP9_F.2: 19.3 ng/ul(97.4nM) x 30ul;
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