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==Mme I digestion== 1 units per 1ug phiX 174 DNA 1 phiX = 5 sites/5389bp 1 amplicon = 2 sites/300bp 7.2 units MmeI to digest 1ug capure PCR amplicons => 2.16 units to digest 300ng. VOL REAGENT Conc Final 30.6 ul DNA + H2O ~300ng 4.0 ul NEBuffer 4 10x 1x 2.4 ul MmeI 2 units/ul 4.8 units (add double amount of required MmeI) 3.0 ul SAM 1mM 75uM -------------------------------------------- 40.0 ul TOTAL Incubate at 37C for 1 hr. Purify with 1 Qiaquick column each, elute with 30ul EB. Run 1 ul each on 6% TBE gel [[File:ZhangLab 2 2010-08-25 JH-tumor 499 536 MmeI digest.png |300px]] Expected digest size: 300-43-45=212bp. '''Quantification with low mass ladder''' {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Signal Intensity''' | align="center" style="background:#f0f0f0;"|'''Volume Loaded''' | align="center" style="background:#f0f0f0;"|'''Calculated amount DNA(ng)''' |- | 0.1x low mass ladder||8966.52||3||0.75 |- | 0.1x low mass ladder||15987.17||6||1.5 |- | 5T partially digested||5545.29||1||0.38 |- | 5T fully digested||2309.56||1||bad |- | 5N partially digested||2578.99||1||0.07 |- | 5N fully digested||974.66||1||bad |- | 4T partially digested||5612.13||1||0.39 |- | 4T fully digested||4521.19||1||0.28 |- | 4N partially digested||912.56||1||bad |} 536T -> 80% of partially digested band, 0.38ng/1ul*0.8*20 = ~6ng 536N -> 80% of partially digested band, 0.07ng/1ul*0.8*20 = ~1ng 499T -> 0.28*20 = ~6ng 499N -> est. = to 536N based on gel = ~1ng
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