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==Perform another PCR on the remaining PAGE size selected DNA (04/10/08)== Template 5ul 2x Taq Master mix 200 100uM AmpF6.2SolH 1.6ul 100uM AmpR6.2SolH 1.6ul 1mM dUTP 4ul 50X SYBG I 3.2ul H2O 172ul split into 4x100ul 94C 2min -> (94C 30S -> 58C 30sec -> 72C 20S) x 8 -> 72C 3min -> 15C hold. Mix all 4x100ul amplicon, purified with Zymo Concentrator-25. ===Make shotgun sequencing library=== ====Mme I digestion==== Qiaquick purified DNA 26ul x 34ng/ul 10X NEBuffer 4: 6ul 1mM SAM 2ul Mme I (2U/ul) 5ul H2O 21ul 37C 90min -> 85C 20min -> 4C hold. MinElute purification -> 6% TBE PAGE size selection -> the DNA migration pattern is still not right I think it is due to the 85C head inactivation step, which denatured DNA. So what I was see are ssDNA or partially annealed ds/ssDNA. [[Image:2008-04-11-Cpg-Jurkat-2nd-PCR-MmeI-cut.png|300px]]
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