Experiments | CSMU-Taiwan

Preparing competent cells

1. Select a single E. coli colony, strain BL21 DE3, cultured in 3 ml Luria-Bertani(LB) culture solution, placed in a 37˚C incubator 160 rpm oscillation for 16~20 hours.
2. Take 1ml overnight culture solution into 100ml LB culture solution (1:100), placed in a 37˚C incubator 160 rpm oscillation for 3~4 hours, until the measured light absorption value of 0D600 in the range of 0.45~0.6.
3. After cultivation, collect all the bacterial liquid to 50ml centrifuge tubes which can be divided into two tubes and place it on the ice for 10 minutes.
4. Then centrifuge the bacterial liquid completed by the ice bath, set the rotation speed of 3000 rpm, the temperature of 4°C, and the time is 10 minutes.
5. After centrifugal, remove the supernatant, leave the bacterial sediment, add 100 ml of trituration buffer (100 mM CaCI2, 10mM MgCl2, 40 mM sodium acetate, pH 5.2), and pipetting until the bacteria complete suspended. Centrifuging to collect bacteria, set the rotation speed of 3000pm, 4°C, 10 minutes.
6. After centrifugal, remove the supernatant and leave the bacterial sediment. Repeat step 5, add 100 ml of trituration buffer, and absorb the sediment repeatedly with pipetting to make the bacteria completely suspended and leave it on the ice for 45 minutes.
7. Then put the centrifuge to centrifuge and set it to 3000 rpm and 4°C for 10 minutes.
8. Remove the supernatant, leave the bacterial sediment, add 2 ml trituration buffer, and pipetting the sediment. Mix to make it fully suspended, that is, to complete the preparation of competent cells.
9. If it needs to be stored, can add 80% glycerin slowly and evenly, so that the final concentration of glycerin in the bacterial liquid is 15~25%, and then the competent cells are packaged in 100µl/ tube and stored at -80°C for backup, but the longer they are placed, the less efficient they are, so they need to be used for at least 14 days.

Heat shock

In a sterile operating table

1. Preheat the dry bath to 42°C.
2. Put the competent cells in an ice bucket.
3. Add 50 µl of competent cells and 2 µl of plasmid (avoid pipetting; draw a circle on the Eppendorf wall)
4. Incubate on ice for 30 minutes.
5. Place the mixture into the dry bath for 90 seconds.
6. Return it to ice for 5 minutes.
7. Add 800 µl of LB media.
8. Incubate in a 37°C shaking incubator for 1.5 hours.
9. Incubate LB-agar plate containing Kanamycin at 37°C overnight.

Cultivate a small quantity of bacteria

1. Begin by warming Kanamycin (KM) until it is completely melted. Vortex thoroughly and centrifuge quickly once it has all melted.
2. Using an auto pipette, aspirate 4 ml of LB into a culture tube.
• Ensure the mouth and cap of the LB bottle are flame-sterilized when opening or closing.
• Place used glass tips from the auto pipette back into their original packaging before disposing of them as infectious waste.
3. Add 4 µl of kanamycin (antibiotic), which is calculated as 0.001 times the volume of LB.
4. While adding, ensure that you penetrate the liquid surface, as the quantity is minimal, and carefully observe whether it has been completely added.
5. Collect bacteria from the plates and add them to the culture tube, mixing by pipetting.
6. Close the culture tube.
7. Place it in a tissue culture rotator and cultivate for 16-20 hours (at a speed of approximately 100 rpm).

Scale up the culture

1. Begin by adding 200 ml of LB and 200 µl of KM to a conical flask.
2. Add 3 ml of bacterial culture from the culture tubes.
3. Place it in a shaking incubator with the following settings:
        • Incline the flask.
        • Set the speed to 200 rpm.
        • Ensure there is no noise inside the incubator when the desired speed is reached.
4. After 2 hours, remove and measure the OD value. If the OD value has not reached the desired range, measure it every hour.
5. When the OD value is between 0.6 and 0.8, take 1 ml of the culture and transfer it to a centrifuge tube.
        • Label the tube with "BI" and the bacterial strain name.
        • Centrifuge for five minutes.
        • Discard the supernatant.
        • Store at -20°C (inside the iGEM box).
6. Add 200 µl of IPTG to the conical flask.
7. Start a timer for 4 hours.

Separate bacteria from the culture

1. Transfer 1 ml to a centrifuge tube:
        • Label the tube with the strain name "AI.”
        • Centrifuge for five minutes.
        • Discard the supernatant.
        • Store in a -20°C freezer (inside the iGEM box).
2. Pour the remaining bacterial suspension into a high-speed centrifuge bottle labeled "Bottle 41."
3. Adjust the weight of all centrifuge bottles to be equal.
4. When using the high-speed centrifuge machine, ensure the following:
        • Verify that the rotor and the interior of the machine are free of moisture (if present, wipe clean with lens paper).
        • Securely close and place the bottles symmetrically.
        • Set the rotor number to 41.
        • Confirm that the rotor number matches the machine.
        • Set the speed to 7000 rpm, time to 8 minutes, and temperature to 4°C.
5. After centrifugation, discard the supernatant and store in a -20°C freezer.

Cell Disruption

Ensure to take 'before & after' photos

1. Use an autopipette to draw 20 ml of lysis buffer and resuspend the pellet (vortex the entire bottle).

2. Transfer the bacterial solution into a 50 ml centrifuge tube and place it in an ice bucket.
3. Prepare the following:
        • ddH2O
        • Kimwipes
        • 70% ethanol
        • Probeß
        • Ice bucket with centrifuge tube
4. Clean the probe with ethanol, followed by ddH2O, and finally wipe it dry with Kimwipes.
5. Insert the probe vertically into the centrifuge tube, taking care to ensure that it does not touch the bottom or sides of the tube.
6. The centrifuge tube should be placed on ice, ensuring that the ice level is above the bacterial suspension.
7. Start the disruption process:
        (1) Power on [I]
        (2) Set time to 6 minutes.
        (3) Pulse on for 10 seconds
        (4) Off for 20 seconds
        (5) Set amplification to 60%
        (6) Total duration: 18 minutes
8. During the process, monitor the ice level, adding more ice during the 'off' phase if it melts. If the bacterial suspension is not clear enough, you may repeat the process.
9. After completion, retrieve the centrifuge tube and clean the probe with ethanol, followed by ddH2O, and then wipe it dry with Kimwipes.
10. Transfer the disrupted bacteria into a high-speed centrifuge tube, and balance with ddH2O in another tube.
11. Perform high-speed centrifugation to separate bacterial debris and proteins:
        • Rotor: 46
        • Speed: 12,000 rpm
        • Duration: 10 minutes
        • Temperature: 4°C
12. After centrifugation, collect 1 ml of the supernatant (labeled as 'sup.') and store it in a -20°C freezer.

His Tag

1. Add ddH2O into the column to check the flow rate. If there is any blockage, lift the top cap and gently tap it a few times.

2. Vortex the entire Ni-NTA agarose, then add 1 ml into the column.
3. Rinse with ddH2O 1-2 times to remove ethanol.
4. Fill the column with PBS, repeating this step twice.
5. When PBS is close to the resin, plug the column with a stopper.
6. Pipette 1 ml of supernatant into the column.
7. Mix well, then draw the supernatant back (leaving a small amount in the column).
8. Allow binding for 1-3 hours.
9. Add a small amount of lysis buffer to the column and place it in the refrigerator.
10. After the designated time, set up the column, and pour the pre-bound protein in batches into the column (collect in 50 ml centrifuge tubes).
11. Add lysis buffer to the bottle containing the centrifuge tubes and pour it into the column in batches (wash).
12. Finally, add 2 ml of elution buffer (starting with low concentration and gradually increasing) to the column.

13. Collect 1 ml in each of the ten centrifuge tubes.
14. Store at -20°C. (All procedures must be performed in the refrigerator)

Protein Quantification

1. Prepare a tube containing a 1 mg/ml BSA solution.
2. Prepare five different concentrations of BSA solutions as indicated in the figure below (mix thoroughly by vortex).

3. Dispense 10 µl of both the well-mixed BSA solutions and the protein of interest into a 96-well plate.
4. Add 200 µl of Bio-Red to each well (this step should be performed in the absence of light).
5. Incubate for 15 minutes.
6. Measure the absorbance at 595 nm using a spectrophotometer.

SDS Page

1. According to the configured amount, add 5x dye and protein sample to a 1.5 ml Eppendorf, and write the name of the protein.
2. After mixing, use thermomixer comfort, heat it to 95 degrees, time it for five minutes, then put it in an ice bucket and time it for five minutes.
3. After complete gelation of the stacking gel, taking out the comb. Take the glass plates out of the casting frame and set them in the cell buffer dam.
4. Fill inner chamber with running buffer and pour the buffer to the required level in the outer chamber.
5. Load prepared samples into wells and make sure not to overflow. Then cover the lid with power cables. (Confirm that the positive and negative poles of the lid and power supply are paired correctly)
6. Running 70V voltage for half hours, then running 90V voltage for 2 hours.
7. When the dye front has nearly run off the bottom of the gel, turning off the power source.
8. Pry apart the gel casing using the green lever tool, put gel into a empty box.
9. Immerse the gel with staining solution(0.3 % Coomassie Brilliant Blue R-250 (w/v), 45 % Methanol (v/v), 10 % Glacial acetic acid (v/v), 45 % ddH2O), and slowly shake it on horizontal rotator for about 20-30min.
10. Immerse the gel in distaining solution(20%Methanol (v/v), 10% Glacial acetic acid (v/v), 70% ddH2O) and put it on the horizontal rotator for about 20-30min.
11. Change the distaining solution for 3-5 times until you can see clear bands with almost no blue background.

Experiments (Protocol)