Abstract: At present, the preparation of competent cells is usually prepared by ice-cold CaCl2 treatment of bacteria, that is, the low-permeability CaCl2 solution is used to treat the rapidly growing bacteria at low temperature (0 ° C), thereby obtaining competent bacteria. The key of the method The selected bacteria must be in the logarithmic growth phase, and the experimental operation must be carried out at low temperatures. Principle:
At present, the preparation of competent cells is usually prepared by ice-cold CaCl2 treatment of bacteria, that is, the low-permeability CaCl2 solution is used to treat the rapidly growing bacteria at low temperature (0 ° C), thereby obtaining competent bacteria. At this time, the bacteria expand into a spherical shape. Under these conditions, the exogenous DNA molecule is easy to form an anti-DNase hydroxy-calcium phosphate complex, which adheres to the surface of bacteria and promotes the absorption of DNA by heat shock. The transformation efficiency can reach 106-107 transformants/μg DNA. .
The key to this method is that the selected bacteria must be in the logarithmic growth phase, and the experimental operation must be carried out at low temperatures.
operating:
1. Take the frozen strain at -70 °C, inoculate the bacteria in the culture dish by scribing, mark well, and incubate at 37 °C overnight.
2. On the next day, pick a single colony from the plate, inoculate it into a test tube containing 3 ml of LB medium, and incubate overnight at 37 ° C. Inoculate 1 ml of the next day inoculum into a 500 ml flask containing 100 ml of LB medium, 37 °C violent shaking culture for about 2~3 hours (200~300r/min)
3. When the colony 600nm OD value reaches 0.3-0.4, the flask is taken out and placed on ice for 10-15 minutes. Under sterile conditions, the bacterial solution is poured into a 50 ml centrifuge tube. 4 ° C, 4000 g centrifugation for 10 minutes.
4. Discard the supernatant, pour the tube on the dry filter paper for 1 min, and blot the remaining culture solution. Add 10 ml of 0.1 M CaCl2 to the centrifuge tube, mix by shaking, suspend the cells, and ice bath for 30 minutes.
5, 4 ° C, 4000g centrifugation for 10 minutes, discard the supernatant, pour the tube on the dry filter paper for 1min, blot the remaining culture solution. Add 4ml ice pre-cooled 0.1M CaCl2, resuspend the bacteria. 0.2ml per tube Packing, storage at 4 ° C, use within 24-48 hours. If not used temporarily, can be stored in a low temperature refrigerator at -70 ° C.
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