Materials and reagents
Constant temperature rotary shaker
2. Flask flask (capacity 1 or 2L)
3. 50ml sterile centrifuge tube
4. Low temperature high speed centrifuge
5. SB medium
Trypsin for bacterial culture 20g
Yeast Extract for Bacterial Culture 5g
NaCl 0.5g
Deionized water 950ml
250mmol/L KCl solution 10ml
The pH of the solution was adjusted to 7.0 with 5N NaOH, deionized water was added to 1 L, and autoclaved.
6. 20% glucose solution
7. 1mol/L MgCl 2 solution
8. 10% glycerin
Steps
1. Pick a TG1 clone from fresh LB plate culture and inoculate in 10 ml SB medium.
2. Incubate overnight at 37 °C on a shaker.
3. Transfer 2.5 ml of the culture to 0.5 L SB and add 10 ml of 20% glucose and 5 ml of 1 mol/L MgCl 2 .
4. Incubate at 37 ° C until OD600 = 0.7 to 0.8.
5. The culture was placed in an ice bath for 15 min, then centrifuged at 4000 r/min for 20 min at 4 ° C, and the supernatant was discarded.
6. The bacteria were suspended in 1/2 volume of pre-cooled 10% glycerol, centrifuged at 4000 r/min for 20 min at 4 ° C, and the supernatant was discarded.
7. Repeat step (6).
8. Suspend the bacteria in 15 ml of 10% glycerol and transfer to a 50 ml centrifuge tube. Centrifuge at 3500 r/min for 15 min at 4 °C. The supernatant was carefully removed to obtain 4 to 5 ml of bacteria. Quickly hang it and resuspend;
9. Dispense into 200 μl or 40 μl aliquots and perform in an ethanol/dry ice bath. Store at -70 °C.
10. The conversion efficiency of competent bacteria was tested using the pUC19 plasmid and should reach 10 9 cfu/μg DNA.
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