Rb/Cacl Transformation Salts, Used With Super-Comp Media, Each

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Super-comp is an extremely rich medium that significantly increases the competency of the cells when using a simple rbcl/cacl means of preparing competent host cells.Dilute 1ml of a fresh overnight culture of host cells grown in lb medium into 25ml of 37°c super-comp media in a 500ml flaskincubate cells with vigorous aeration for 1-2 hours until cells reach mid-log phase (approximately 8 x 108 cells/ml, od600 reading 0.8 For most e. Coli host cells)centrifuge culture in a sterile tube at 4°c to pellet cells. Resuspend pellet in 15ml of ice cold 1x rbcl/cacl transformation saltsafter 15 min. On ice, centrifuge to pellet cells and gently resuspend in 2ml of ice cold 1x rbcl/cacl transformation salts; keep cell suspension on icecells are now competent and are immediately ready for dna uptake. For maximum transformation efficiency, store for 12-24 hours at 4°c to increase competency 4-6 fold (at 48 hours they will be approximately as efficient as when first made). Competent cells can also be frozen in aliquots at -80°c after adding glycerol to 20%.