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Tuesday, November 18, 2003
Description Quick transformation Procedure Take 0.5 ml of culture and spin 10 sec in microfuge. Decant the tube by inverting it and shaking it once. Alternatively, one can pick a colony (2-3 mm in diameter) from a plate with a toothpick and transfer cells to sterile 1.5 ml microfuge tube (as long as the plate is not dried out, colonies can be used from plates stored in the fridge for 3 months, maybe more). Add 10 l of carrier DNA (100 g) plus 1 g transforming DNA(in 10 l) and vortex well. (Carrier DNA does not need to be added if the transforming DNA has come from mini-prep DNA which has not been RNased). Add 0.5 ml PLATE solution and vortex. Add 57 l DMSO and vortex briefly. Leave for 15 min at RT. Heat shock for 15 min at 42 deg C. Pellet cells in microfuge for a few seconds at 10 krpm. Carefully remove supernatant. Add 200 l TE to the cell pellet and gently resuspend cells by aspirating up-and-down with a pipette tip. Immediately spread suspended cells onto selective plates. NOTES - the yield of transformants increases linearly up to about 100-200 g of transforming DNA. - the optimal number of cells per transformation is about 2E8 cells/ml. Cells + DNA volume should be about 140 l. In other words, PLATE:(Cells + DNA) should be about 3.5:1. Recipes 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) |
Quick transformation
时间:2005-07-18 00:00来源:Scienceboard.net 作者:admin
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