for making competent cells to transform into

we used the following protocol

harvesting protocol

Purpose: Generate crude extract from E. coli cell pellets using sonication

1. Obtain cell pellets from -80 freezer and fully submerge them in ice. Let your samples thaw between 1 to 2 hours. In the meantime, make S30 buffer minus DTT.
2. Once your samples are ready, add fresh DTT to your S30 buffer. Add 0.8 mL of S30 buffer/g of rEcoli pellet or 1 mL of S30 buffer/g of BL21(DE3)* pellet.
3. Vortex samples using 15 sec bursts followed by at least a 30 sec cool down in ice in between each burst. Typically, 10 to 15 vortex bursts are required to fully resuspend your pellets in S30 buffer.
4. Once your samples are fully resuspended, let them rest for at least 5 min to allow for the level of cell/buffer mixture to rise. In the meantime, prepare your ice/water bath in a baked glass beaker.
5. Once the level of your cell/buffer mixture has stopped rising, feel free to combine samples if necessary for your experiments.
6. Using your P1000 pipette, aliquot out 2 x 700 µL of cell/buffer mixture to a new prechilled and labeled 1.5 mL microfuge tube. You may make multiple samples if you have enough material.
   Typically, 4 g of cells produces about three 1.4 mL samples
7. Prep the sonicator by taking off the yellow probe tip protector and cleaning the probe with 70% ethanol followed by nanopure water. Dry using a Kim Wipe.
8. Turn on the machine and set the amplitude to 50% and the timing of the pulses to 45 sec on, 59 sec off. The cycle should be repeated 3 times with your samples in the ice water bath and should produce about 942 J total.
   Keep the probe ~¾ into your sample and be sure to swirl your sample around using your hand to ensure maximum lysis efficiency. Note, this step requires some technique and may take a few tries to perfect.
9. Add 3 µL of DTT for every 1 mL of sonicated extract immediately after processing your samples. Typically, 4 µL is added to a 1.4 mL sonicated sample. Sharply invert the tube with DTT between 4 to 8 times to mix.
10. Once you have sonicated all of your samples, centrifuge your samples at 12,000Xg for 10 min at 4˚C. Set up new 1.5 mL microfuge tubes to collect the supernatant. The pellet obtained will consist of unlysed cells followed by a layer of large membrane fractions.
11. BL21(DE3)* Samples: There is no need for a runoff reaction to improve activity of these sample, thus the supernatant resulting from step 10 is sufficient for generating crude extract. Stop here for preparation of BL21(DE3)* extract.
12. Centrifugation of protein aggregates can be performed at 10,000Xg for 10 min at 4˚C. Take out the top 500 µL of the supernatant and store it however you feel is appropriate for your experiments. Typically, 500 µL gives the highest activity and is sufficient to avoid the pellet. Feel free to also combine and speparate out extracts at this point for different experiment purposes.
    I normally take out ~30 µL of sample to perform preliminary testing prior to fully aliquoting out the extract.
13. Flash freeze your samples in liquid nitrogen (extract quality is good for up to 3 freeze/thaws) and store in the -80˚C freezer.
14. Measure protein concentration using a Bradford assay. Typical concentrations observed are between 30 to 50 mg/mL

Key parameters for optimizing extract performance (Rey’s opinion):

• OD (~3) and pH (>6.9) of harvest point
• Minimizing time of processing cell pellets during harvest (faster is better)
• Time of sonication (color change observed should be very similar to figure 1)
• Volume of supernatant taken at each step during extract preparation
• Temperature of cells and lysate (the colder the better) => always keep in ice when possible