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===dA-tailing=== (NEB) '''x 14rxn mix''' End repaired DNA 37ul - dA-tailing buffer, 10x 5ul 70ul Klenow Fragment 3ul 42ul H2O 5ul 70ul ------------------------------------------- - aliquot 13ul of reaction mix into PCR tube Total 50ul - add 37ul of end repaired DNA - incubate in thermomixer at 37C for 30 minutes, purify with 0.7x AMPure beads, elute with 40ul EB. - quantify each reaction with Nanodrop and PAGE quantification. [[File:ZhangLab_2 2010-10-25 12hr 18min_MG SAGs no1,2,4,5,8,9,12,13,14,15 dAtailed.jpg|500px|Image: 500 pixels]] [[File:ZhangLab_2 2010-10-25 12hr 18min_MG SAGs no16,32,ntc dAtailed.jpg|220px|Image: 220 pixels]]<br> <br>Figure1. LM ladder 6ul, LM ladder 3ul, MG1, MG2, MG4, MG5, MG8, MG9, MG12, MG13, MG14, MG15<br> Figure2. LM ladder 6ul, LM ladder 3ul, MG16, MG32, NTC '''Sample ID''' '''Conc.(ng/ul)by Nanodrop''' '''Conc.(ng/ul)by PAGE quantification''' MG1 -0.1 1.66* MG2 2.6 8.05* MG4 3.2 9.77* MG5 2.3 5.04* MG8 0.8 0.99** MG9 0.6 3.18* MG12 0.2 - MG13 0.3 0.14** MG14 -0.5 0.27** MG15 -0.0 2.62* MG16 -0.2 0.73** MG32 0.3 1.35* NTC 0.5 - -From DNA conc. by PAGE quantification, DNA samples were divided in two subgroups - '''Gr.1 (*) Conc.>1.0ng/ul:''' MG1, MG2, MG4, MG5, MG9, MG15 & MG32 - '''Gr.2 (**) Conc.<1.0ng/ul:''' MG8, MG13, MG14 & MG16 - Sample MG12 didn't show the product in the gel then it was repeated again with larger amount of sheared DNA, 65ul in end repaired step. *** After Nanodrop check, it was grouped into Gr.2
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