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.
9. 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.