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== Colony PCR == - Lysed the cells in 10 ul H2O by using 10ul pipette tip to pick up the white single colony and pipette up and down in the 96-well plate containing 10ul H2O - heated at 98C for 10 min - vortexed vigorously - put the tube on ice box and spin down the cell lysis {| {{table}} border = 2 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''84 rxn mix''' |- | Cell lysis||2.00||0.00 |- | 10uM Syb_FP5A||1.00||84.00 |- | 10uM Syb_RP7||1.00||84.00 |- | 2X Taq MM||25.00||2,100.00 |- | H20||21.00||1,764.00 |- | Total||50.00||4,200.00 |} * Aliquot 48ul, add 2ul of cell lysis DNA template '''Program''' 96C 3min -> (95C 30s -> 58C 45s -> 72C 45s)x36 cycles --> 72C 5min --> hold at 15C Note: Always get evaporation problem of the samples in the edge of the 96-well plate (long time amplification ~3hr) even sealing very well '''2012_06_04''' - Analyzed in 2% E-gel by loading PCR product 10ul [[File:ZhangLab_2 2012-06-04 10hr 44min_EGel1.jpg| 300px]] - Select 20 positive clones from each USER volume (later) - Purified with 1vol. AmPure beads and eluted with 30ul EB buffer - Measured DNA conc. by Nanodrop
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