Preparation of electric shock and heat shock competent cells
Experimental reagent
LB medium, KB medium, 10% glycerol, liquid nitrogen, 0.1M CaC1 2 solution
Laboratory equipment
5毫å‡çš„离心管, Ultra-clean workbench, shaker, centrifuge, 250mL centrifuge bottle, 0. 5mL centrifuge tube
Experimental Materials
Escherichia coli, Agrobacterium and Pseudomonas
Experimental procedure
1. Preparation of electroporation competent cells
1) -80 °C Preserved Escherichia coli, Agrobacterium or Pseudomonas on solid plates (DH5a, BL21 on LB plates, GV3101 on LB/Gen; Ps pv. tomato 0288-9 and P. syringae pv. tabaci 6505 on the KB plate) scribing.
2) Inoculate the monoclonal in 5-10 mL of liquid medium, culture overnight at 37 ° C (E. coli) or 28 ° C (Agrobacterium and Pseudomonas).
3) Transfer to 500-1000mL liquid medium at 1:100-1:500, culture at 37 °C for 3-4 hours (E. coli) or culture at 28 °C for 8-10 hours (Agrobacterium and Pseudomonas transformation) to The bacteria reach the logarithmic growth phase.
4) The bacterial solution was placed in a 250 mL centrifuge bottle, placed on ice for 30 minutes, and centrifuged at 4 ° C for 15 minutes.
5) Discard the supernatant, add 200 mL of 10% glycerol, slowly shake the bacteria on ice, and centrifuge at 15 °C for 15 minutes.
6) Repeat the previous step 2-3 times, finally discard the supernatant and add a small amount of 10% glycerol to resuspend the bacteria.
7) Dispense in a 0.5 mL centrifuge tube, 40ul per tube, liquid nitrogen frozen, stored at -80 °C.
2. Preparation of heat shock competent state
1) Pick up the E. coli BL21 monoclonal inoculate into 5 mL of LB medium and incubate overnight at 37 °C with shaking.
2) Transfer to 500-1000 mL of liquid medium at 1:100-1:500 and incubate at 37 °C for 3-4 hours until the bacteria reach logarithmic growth phase.
3) The bacterial solution was placed in a 250 mL centrifuge bottle, placed on ice for 30 minutes, and centrifuged at 4 ° C for 15 minutes.
4) Discard the supernatant, gently suspend the cells with 20 mL of pre-cooled 0.1 M CaC1 2 solution, place on ice for 15-30 minutes, and centrifuge at 4 ° C for 5 minutes.
5) Discard the supernatant, add 2 mL of ice-cold 0.1 M CaC1 2 solution, and resuspend the cells.
6) Dispense in a 0.5 mL centrifuge tube, 40ul per tube, liquid nitrogen frozen, stored at -80 °C.
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