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Tuesday, November 18, 2003
Description PLATE Transformation Procedure COMPETENCY: Inoculate a single colony into 100 ml YPD (or selective media if necessary). Grow on shaker to O.D.(600) = 1.0 (O.D.(600) between 0.6 and 1.8 is fine) at 30oC overnight. Spin down cells in table top centrifuge at 2,000 rpm. Resuspend cells in 10 ml TEL. Shake vigorously overnight at room temperature; you can skip this step and proceed to the next if you don't want to save cells. Spin down cells and resuspend in 1 ml TEL. Use for transformation. Cells can be kept in the refrigerator and used for up to a month. TRANSFORMATION: Add 50 ug salmon sperm DNA (usually 5 ul of 10 mg/ml) to 100 ul of competent cells in a sterile 1.5 ml tube. Add DNA to suspension. Typically 1 ug of qiagen DNA, or 5 ul of miniprep DNA for uncut plasmids. Use more for integrating constructs. Incubate 30 minutes at room temperature. Add 0.7 ml PLATE solution, resuspend thoroughly. Incubate 1 hour at room temperature. Heat shock at 42oC for 5-10 minutes. Plate on appropriate selective plates. Recipes Necessary Solutions: 1X TEL buffer 10 mM Tris-HCl, pH 7.5 1 mM EDTA 0.1M LiAc PLATE solution 40% PEG 3350 0.1M LiAc 10 mM Tris-HCl, pH 7.5 1 mM EDTA Supplies Tips (责任编辑：泉水) |
PLATE Transformation
时间:2005-07-18 00:00来源:Scienceboard.net 作者:admin
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