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==Exp.1 Library construction on the new capturing reaction started on 01/16/2008== ===Mme I digestion on the 2nd round PCR amplicon=== 500bp+ 2nd PCR all size 2nd PCR 50ul 50ul 32mM SAM 0.5ul 0.5ul Mme I 2U/ul 4ul 4ul 37C 1h -> 75C 20min -> 4C hold. Purify with Bioneer columns, then PAGE size selection. Split the purified DNA into two tubes, one for USER digestion and the other for SolN6 primed synthesis. ===USER enzyme digestion=== Add 2ul USER enzyme into 18ul of DNA in EB buffer, 37C 1h. ===End repair=== Set up end repair reaction (with NEB quick blunting kit) USER DNA 19ul 10X buffer 2.5ul 1mM dNTP 2.5ul Enzyme mix 1ul RT for 30min -> 75C 10min ===SolN6 primed synthesis=== SolF/Rn6 DNA 10ul 10X Klenow buffer 5ul 100uM SolFn6 1ul 10mM dNTP 2ul H2O 30ul 94C 2min -> 16C 1min -> add 1ul 20U/ul Klenow exo- -> 16C 10min -> 37C 10min -> Clean up the reaction with G25 column, then add 6 ul 10X buffer, 2ul dNTp, 1ul 100uM SolRn6-> 94C 1min -> 16C 1min -> add 1ul 20U/ul Klenow exo-, 1ul T4 DNA pol-> 16C 10min -> 37C 10min -> purified with MinElute columns, eluted with 10ul EB. ===Ligation=== USER DNA 15ul 50uM Solexa_1 2ul 50uM Solexa_2 2ul 2X Quick ligation buffer 20ul Quick ligase 1ul mix well -> room temperature for 20min -> 75C 10min Purified with MinElute columns. ===Nick-translation=== DNA 15ul 10X ThermoPol Buffer 2ul 10mM dNTP 0.4ul 1mg/ml BSA 2ul 8U/ul Bst pol 1ul 65C 25min -> place on ice ===PCR=== Nick-translated DNA 5ul SolN6 primed DNA 5ul 2X iProof master mix 25ul 25ul 100uM Solexa_PCR_up 0.25ul 0.25ul 100uM Solexa_PCR_lo 0.25ul 0.25ul H2O 19.3ul 19.3ul 50X SYBG I 0.2ul 0.2ul 98C 30sec -> 18 cycles of (98C 10sec -> 65C 20 sec -> 68C 30sec) -> 72C 3min ->15C hold
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