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Kun:LabNotes/CpgSeq/2008-3-18
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==Reamplify the PAGE size selected Zymo, dNTP 50uM for Solexa sequencing (04/10/08)== Template 5ul 10X Platinium Taq buffer 40ul 50mM MgCl2 12ul 100uM AmpF6.2SolH 1.6ul 100uM AmpR6.2SolH 1.6ul 10mM dNTP 8ul 50X SYBG I 1.6ul H2O 326ul iTaq(5U/ul) 8ul split into 4x100ul 94C 2min -> (94C 30S -> 58C 30sec -> 72C 20S) x 9 -> 72C 3min -> 15C hold. Mix all 4x100ul amplicon, purified 100ul with Qiaquick, 100ul with Zymo Concentrator-25. Qiaquick: 22ng/ul x 30ul Zymo: 28ng/ul x 15ul Clonewell size selection on the Qiauick purified DNA. Purify the remaining 200ul amplicon with Qiaquick, 34ng/ul x 30ul ===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 -> 80C 20min -> 4C hold. I ran the digestion in 6% PAGE gel without purification, it turned out that the DNA migration pattern was totally off, probably due to DNA:protein interaction.
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