1. Preparation of electroporation competent cells

1. Pick a monoclonal colony with a pipette tip and place it in a 50 ml centrifuge tube containing 10 ml of LB liquid medium. (At the same time, the blank of the medium and the tip of the gun)

2.37 ° C, 220 rpm, culture for 14-16 hours.

3. On the next day, pour 10 ml of the bacterial solution into 1000 ml of LB liquid medium at a ratio of 1:100, shake at 37 °C, 220 rpm for 2-3 hours, and measure OD every half hour. When the OD value reaches 0.3. At -0.4, the culture was stopped.

4. The bacterial liquid was pre-cooled on ice for 30 minutes, and then the bacterial liquid was dispensed into a 500 ml pre-cooled centrifuge cup, and centrifuged at 2500 rpm for 10 minutes at 4 °C.

5. Discard the supernatant, add a small amount of ddH ₂ O to the centrifuge cup, suspend the pellet, then fill the centrifuge with water, and centrifuge at 4000 ° C for 10 minutes at 4 ° C.

6. Discard the supernatant, add a small amount of sterilized water, resuspend the cells, and then fill the centrifuge with water, 4000 rpm, 4 ° C, and centrifuge for 10 min.

7. Discard the supernatant, add a small amount of 10% glycerol to the centrifuge cup (sterilized, pre-cooled), resuspend the cells, then add 10% glycerol, 4 ° C, 4000 rpm, centrifuge for 10 min.

8. Discard the supernatant, add 5 ml of 10% glycerol to each centrifuge cup, and suspend the pellet. The bacteria solution was placed in a 1.5 ml centrifuge tube at 300 ul/tube and stored in a -80 °C refrigerator. At the same time, 100 μl of competent state plus 0.01 ng puc18 was directly electroporated to detect transformation efficiency.

9. Observe the growth of transformants the next day and record.

Second, the connection product purification

1. Transfer the ligation product to a 1.5 ml Eppendorf tube and add the following reagents:

10μl of ddH ₂ O

2μl of 3M NaAC (PH5.2)

50μl of absolute ethanol

Mix gently, centrifuge slightly and place at -20 ° C for more than 1 hour;

Centrifuge at 2.4 ° C, top speed for 30 minutes;

3. Carefully remove the supernatant to avoid contact with the sediment at the bottom of the tube;

4. Add 500μl of 70% ethanol, gently invert several times to wash the precipitate (Note: do not mix by centrifugation);

Centrifuge at 5.4 ° C for 5 minutes;

6. Carefully remove the supernatant and place the Eppendorf tube in air until there is no ethanol odor;

7. Add 10 μlddH ₂ O to redissolve the precipitate, store at 4 ° C for short-term storage, and store at -20 ° C for long-term storage;

Third, electricity conversion

1. Remove the competent cells from the -80 ° C refrigerator and thaw on ice;

2. 1 μl of the purified plasmid was placed in a 1.5 ml centrifuge tube and placed on ice with a 0.1 CM electrode cup for pre-cooling.

3. Transfer 40 to 100 ul of thawed competent cells to this 1.5 ml centrifuge tube, carefully mix and place on ice for 10 min.

4. Turn on the motor, adjust to Manual, and adjust the voltage to 2.1KV.

5. Transfer the mixture to the pre-cooled electrode cup and gently tap the electrode cup to evenly enter the bottom of the electrode cup;

6. Push the electrode cup into the electro-converter, press the pulse button, and after hearing the buzzer, quickly add 1000 μl of SOC liquid medium to the electric shock cup, resuspend the cells, and transfer to a 1.5 ml centrifuge tube.

Resuscitation at 7.37 ° C, 220-250 rpm for 1 hour.

8. Take 20 μl of the transformation product and add 160 μl of SOC plate, place in a greenhouse at 37 ° C, and incubate overnight, and view the transformation results the next day. The remaining bacterial solution was added with 1:1 30% glycerol and then mixed and stored at -80 °C.

Note: Each plate with Amp is evenly coated with X-Gal 80μl, SOC 80μl, IPTG 20 μl.

Fourth, the electric shock cup cleaning process

1. Slightly flush the electric shock cup with water.

2. Soak 25% of the 75% alcohol added to the electric shock cup.

3. Discard the alcohol, then rinse it with distilled water for 2~3 times, then use the 1ml gun to take the ultrapure water and repeatedly blow the electric shock cup more than 10 times.

4. Add 2 ml of absolute ethanol to the electric shock cup and soak for 30 minutes.

5. Discard absolute ethanol and spin the ethanol in a ventilated kitchen.

6. Put the cleaned electric shock cup into the refrigerator at -20 °C for use.

Note: 1. Motor cups used for different samples should be separated;

2. Soak for 30 minutes a week with 1% alcohol.

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