Kun:LabNotes/ExonomeSeq/2008-1-24
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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.
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
N6 primed DNA 10ul 50uM Solexa_1 1ul 50uM Solexa_2 1ul 2X Quick ligation buffer 15ul Quick ligase 1ul H2O 2ul 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