枯草杆菌感触感染态细胞的制备及转化(国外英文资料).docVIP

枯草杆菌感受态细胞的制备及转化(国外英文资料) Preparation and transformation of Bacillus subtilis competent cells 1) the strains were inoculated on LB plates and incubated overnight at 37. 2)?? by inoculating loop connected to a ring on the lawn 5mL at 30 DEG C GM I solution, Manyao bed (125 r/min) cultured overnight. 3) the next day, 2 mL was transferred to 18 mL GM I, and 37 (250 r/min) was cultured for 3.5 h. 4)?? then 10 mL medium transfer step to 90 mL GM II, 37 C Manyao bed after cultured for 90 min 5000 g 10 min was collected by centrifugation. 5) with 10 mL, the supernatant of the original culture medium is gently suspended, and the suspended cell is the competent cell, which can be directly used for transformation. 6) the preservation of the competent cells: add 30% of the glycerin to the final concentration of 10%, mix it into the centrifuge tube (0.5 mL/tube), and then store it at -70 centigrade. 7) when removed, the centrifuge tube is removed and dissolved at 45 DEG C in water bath, and then a proper amount of DNA (1 ug/ml) is added to the 0.5 mL bacterial fluid. 8) at 37 DEG C oscillation (200 r/min), after 30 min insulation, coated with corresponding resistant plates, then incubated overnight at 37, and examined and verified transformants the following day. Preparation reagent of Bacillus subtilis common chemosensory cell [1]? 10 * Minimum salt solution: K2HPO4 70 g, KH2PO4 30 g, (NH4) 2SO4 10 g, (Na3C6H5O7, 2H2O) 5 g, MgSO4, 7H2O, 1 g, dissolved in distilled water, followed by water to 500 mL. [2]? Amino acid solution (prepared according to the gene defect of different strains): 10 mg/mL, stored in brown bottle, sterilized 30 min at 113 DEG C, wrapped in black paper. [3]? GM I solution: 1 x minimum salt solution, 95.6 mL, 20% glucose, 2.5 mL, 5% casein hydrolysate, 0.4 mL, 10% yeast juice, 1 mL, 10 mg/mL amino acid solution, 0.5 mL (50 g/mL). [4]? GM? I solution: 1 x 96.98 mL minimum salt solution, 20% glucose 2.5 mL, 5% mL casein hydrolysate 0.08, 10% yeast extract 0.04