for making competent cells to transform into

we used the following protocol

Day 1:

1. Streak out your strain of interest onto an LB plate with antibiotic. Incubate your plate overnight.
2. Prepare 750 mL of 2xYTP for each Tunair flask. Typically, ~7 mL of 5N KOH to bring 2250 mL of 2xYTP up from ~6.9 to 7.2 pH. See excel spreadsheet.
3. Add a filter cap onto the Tunair flask and foil to prevent major water loss from the autoclave run.
4. Prepare 250 mL of a 7.2% glucose solution separately in a baked flask. Make about 5% more volume than required to account for loss from the autoclave run (e.g., if you need 750 mL, make 800 mL). Note, glucose is autoclaved separately from the base to prevent oxidation.
5. Prepare a 2 L glass bottle filled with nanopure water as well. About 200 mL is required per 1 L culture to be harvested.
6. Perform either a Liquid 40 or 45 autoclave cycle on your 750 mL of 2xYTP in your Tunair flasks, Glucose soutioln, and nanopure water to sterilize.
7. In preparation for making your starter culture for your fermentation, autoclave an empty baffled flask ceach starter culture along with a 1 L centrifuge bottle and cap for each culture you plan to harvest on a PreVac 20 cycle.

Day 2:

1. 16 to 18 hours before the inoculation time for the harvest, add 30 mL of regular LB to your autoclaved 125 ml baffled flask using sterile technique. Add appropriate antibiotics and inoculate one colony of your strain grown overnight from a LB plate to begin your starter culture.
2. Store the rotors to be used during the harvest in a 4˚C fridge to ensure the inside is cool enough for the day of harvest.

Day 3:

1. Go over the quick checklist and ensure that you have all the proper materials needed for the harvest.
2. Add 250 mL of 7.2% glucose using a baked glass cylinder into the 750 mL of 2xYTP to complete the production of 2xYTPG with a final glucose concentration of ~2%.
3. Inoculate your starter culture such that the starting OD is within the range of 0.05 to 0.1 and incubate your samples.
4. When culturing a new strain or under new conditions, it is optimal to check the OD and pH every 20 to 30 minutes.
5. Typically, if IPTG induction is needed, it is done about 2 h before harvesting and around OD 0.4 on the Nanodrop or OD 0.25 on the Genysis. Extrapolating the correct time for induction can be done from growth rate data as measured by the plate reader. This step will require optimization for each strain.
6. As the cultures get closer to the optimal OD to harvest, prepare all necessary materials for the harvest including: S30 Buffer (excluding DTT), chilled rotor and centrifuge, centrifuge bottles, liquid nitrogen, measured and chilled 50 mL conical tubes, and DTT aliquot sitting in ice.
7. If you plan to harvest at a higher OD, you may want to consider spiking in 5N KOH in order to maintain the pH of the culture near 7.0.
8. Once your cultures have reached the proper OD, immediately take your 1 L shake flask containing the culture out of the incubator and pour the contents into the 1 L centrifuge bottles in ice. Balance your 1 L samples either with another 1 L culture or simply water. When you balance, be sure to include the lids and to avoid passing the 1 L mark on the bottles to avoid spillage.
9. Place your sample in the large and chilled JLA 8.1 rotor capable of spinning 1 L cultures. Because it has a slow start, it is best to run the first cycle for 15 min at 5x1000g at 4˚C.
10. During this first spin, fully thaw out and add the DTT to your S30 buffer. Flash freeze any remaining DTT and store in your -80˚C box (it can be reused for the next harvest). Additionally, you can lightly bleach the remainder of your culture in the Tunair flask with 10% bleach and rinse your bottles out with nanopure water and allow them to dry as you harvest.
11. Once your spin is over, dump the supernatant into a large container containing a small amount (~15 mL) of 100% bleach. Before inverting your bottle, wipe the inside walls of the bottle with large Kim Wipes to remove as much of the leftover supernatant as possible. Place the centrifuge bottle containing your cell sample in ice. Repeat for all samples.
12. Using a spatula, add you cell pellet into a 50 mL conical tube. To obtain the remaining cells from the spatula and centrifuge bottle, add 2 mL of S30 buffer in the bottle and use the spatula to mix the cells into solution. Pipette the cell/buffer mixture into the 50 mL conical tube. Proceed with your remaining cell samples.
13. During the second spin, it is best to rinse out the 1 L centrifuge bottles and spatulas with 10% bleach followed by a thorough rinsing with nanopure water. At this point, you can also dump out the bleached supernatant from the first spin into the sink. Cleanup of the rotor used for the first spin is also optimal either now or after the second spin.
14. Once your samples are done spinning, dump the supernatant in the sink and add another 25 mL of S30 buffer to your samples and resuspend as in step 23. This step may take up to 15 min.
15. Repeat the above step.
16. During your third spin, check to make sure the centrifuge used in the first step has fully thawed and cleanup the condensation and any spillage that may have occurred.
17. Repeat.
18. Repeat step 25, but this time you will spin for 10 minutes at 7x1000g at 4˚C.
19. Once your samples are ready, dump the supernatant, and before you invert your tubes, clean the residual supernatant as much as possible using a Kim Wipe.
20. At your bench, use a clean P1000 pipette tip to split the cell pellet in half to increase the surface area of the pellet. This will increase the efficiency of pellet resuspension when making extract.
21. Record the weight of your tubes with the pellet and flash freeze your pellet by dipping the tube into liqhuid nitrogen. Store in the -80˚C freezer.
22. After a few hours or after letting your culturing equipment dry overnight, you can wrap your Tunair flasks with their caps and the 1 L bottles and caps individually with aluminum foil and autoclave on a Prevac 20 cycle. If you plan to harvest at a higher OD, you may want to consider spiking in 5N KOH in order to maintain the pH of the culture near 7.0.
23. Once your cultures have reached the proper OD, immediately take your 1 L shake flask containing the culture out of the incubator and pour the contents into the 1 L centrifuge bottles in ice. Balance your 1 L samples either with another 1 L culture or simply water. When you balance, be sure to include the lids and to avoid passing the 1 L mark on the bottles to avoid spillage.
24. Place your sample in the large and chilled JLA 8.1 rotor capable of spinning 1 L cultures. Because it has a slow start, it is best to run the first cycle for 15 min at 5x1000g at 4˚C.

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