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===Make shotgun sequencing library=== ====Mme I digestion==== DNA 20ul x 29ng/ul 10X NEBuffer 4: 4ul 1mM SAM 1.3ul Mme I (2U/ul) 4ul H2O 10ul 37C 90min 4C hold. Qiaquick purification -> 6% TBE PAGE size selection The DNA migration pattern was still not right. Looks like Mme I didn't make any cut at all. Repeat the digestion by adding 36ul H2O, 5ul 1mM SAM, 4ul Mme I, 5ul 10X NEBuffer 4. 37C 4h. [[Image:2008-04-13-Cpg-Jurkat-MmeI-Size-selection.png|200px]] ====End repair (Epicentre End-it repair kit)==== Resuspended the pallet in 30ul ddH2O, add 3ul USER enzyme, 37C 4h. Add 5ul 10x Buffer, 5ul dNTP mix, 5ul ATP, 1ul Enzyme mix. RT overnight. Purified with MinDlute kit. 16ng/ul x 10ul ====Blunt-end ligation==== 25ng/ul end-repaired DNA 10ul 100uM Solexa_1 pair 0.5ul 100uM Solexa_2 pair 0.5ul 2X Quick ligase buffer 15ul Water 3ul Quick Ligase (NEB) 1ul RT 10min -> Purified with Qiaquick column. PAGE size selection, ethanol precipitation [[Image:2008-04-15-Sol-adaptor-ligation-size-selection.png|300px]]
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